Nucleotide sequences of HIV-1 group (or subgroup) O retroviral antigens

ABSTRACT

An HIV-1 type (or subtype) O retrovirus protein, or a natural or synthetic polypeptide or peptide including at least a part of said protein, which is capable of being recognised by antibodies isolated from a serum resulting from infection by an HIV-1 type O VAU strain or an HIV-1 type (or subtype) O DUR strain.

[0001] The invention relates to the antigens obtained by expression ofnucleotide sequences or by chemical synthesis, for example using AppliedBiosystems brand synthesizers, present in HIV-1 group (or subgroup) Ovariants and more particularly the antigens corresponding to those whichmay be isolated from viral particles. By way of example of HIV-1 virusesof the subgroup O, reference is made to the HIV-1_((VAU)) isolate and tothe HIV-1_((DUR)) isolate.

[0002] The invention also relates to monoclonal or polyclonal antibodiesinduced by these antigens.

[0003] The invention also relates to cloned DNA sequences either havingsequence analogy or complementarity with the genomic RNA of theabovementioned virus. The invention also relates to processes for thepreparation of these cloned DNA sequences. The invention also relates topolypeptides containing amino acid sequences coded for by the cloned DNAsequences.

[0004] Furthermore, the invention relates to applications of theantigens mentioned above to the in vitro detection in at-riskindividuals of certain forms of AIDS and, as regards some of them, tothe production of immunogenic compositions and vaccinating compositionsagainst this retrovirus. Similarly, the invention relates toapplications of the abovementioned antibodies for the same purposes and,for some of them, to their application to the production of activeprinciples for medicinal products against this human AIDS.

[0005] The invention also relates to the application of the cloned DNAsequences and of the polypeptides obtained from these sequences asprobes or primers for gene amplification, in diagnostic kits.

[0006] The invention also relates to antigenic compositions which may beobtained by chemical synthesis or by expression in a recombinant cellhost and which allow the diagnosis of an infection due to a humanretrovirus of HIV type independently of the HIV-1 or HIV-2 subtype. Suchcompositions comprise at least one peptide chosen from the antigenicpeptides common to the HIV-1, HIV-2,HIV-1_((DUR)) and HIV-1_((VAU))viruses or variants of the antigenic peptides possessing similarimmunogenic characteristics.

[0007] The invention is also directed toward compositions which allowthe specific diagnosis of an infection due to a human retrovirus ofHIV-1 type, more particularly HIV-1, group M, HIV-2 or HIV-1 group (orsubgroup) O and comprising at least one antigenic peptide specific forthe HIV-1 virus, an antigenic peptide specific for the HIV-2 virus andan antigenic peptide specific for the HIV-1 group (or subgroup) O virusor variants of these antigenic peptides possessing similar immunogeniccharacteristics. More particularly, the antigenic peptides are derivedfrom the envelope protein of HIV-1 group M and HIV-2 and HIV-1 group (orsubgroup) O viruses.

[0008] The invention is moreover directed toward a peptide allowingdetection of anti-HIV antibodies which the peptides of the prior art didnot always make it possible to detect, based in particular on thediscovery of a new HIV-1 strain: HIV-1 DUR. The antiserum directedagainst it does not always have reactivity with the peptides of theconsensus HIV as it is used nowadays. The term “consensus HIV” refers tothe regions which are conserved between isolates and whose demonstrationis essential to the design of diagnostic reagents or vaccines, and whosemutations impart resistance to antiviral medicinal products. The term“peptide” used in the present text defines both oligopeptides andpolypeptides.

STATE OF THE ART

[0009] Two types of human immunodeficiency virus (HIV) which haveresponsibility for the development of an LAS or AIDS have been isolatedand characterized. A first virus, known as LAV-1 or HIV-1, was isolatedand described in GB patent application 8324,800 and patent applicationEP 84401,834 of Sep. 14, 1984. This virus was also described by F.Barré-Sinoussi et al. in Science (1983), 220, 868-871.

[0010] The type 2HIV retrovirus belongs to a separate class and has onlya limited immunological relationship with type 1HIV retroviruses. HIV-2retroviruses have been described in European patent application No.87,400,151,4 published under the number 239,425.

[0011] The HIV-1 retrovirus is the most common and its presence ispredominant in several regions worldwide. As regards the HIV-2retrovirus, it is most often found in West Africa, although itspropagation outside this region has recently been documented by Grez etal., (1994) J. Virol. 68, 2161-2168.

[0012] The totality of primate immunodeficiency lenti-viruses,comprising the type 1 and type 2 human immunodeficiency viruses as wellas several types of non-human primate viruses, is increasing in size andcomplexity. The most common of these viruses, HIV-1, is currentlyspreading in the form of a worldwide epidemic and is responsible for amajor public health problem. Shortly after the identification andmolecular characterization of this virus, it was recognized as beinghighly variable, and currently comprises several subtypes (Myers, 1994,Louwagie, et al. 1993, Louwagie et al. 1992, Myers, G. (1994) HIV-1subtypes and phylogenetics trees. In: Human Retrovirus and AIDS 1994;Myers, G., Korber, B., Wain-Hobson, S., Smith, R. F. and Pavlakis, G.N., Eds. Los Alamos National Laboratory, Los Alamos, N. Mex.III-2-III-9.). This differentiation of subtypes is mainly based on thedivergence of the gag and env genes. At least 6 subtypes have beenidentified, designated A to F, but several are still likely to emergefrom the ongoing extensive worldwide survey on the isolates of HIV-1. Ithas been found that these various subtypes are equidistant from eachother, in a phylogenetic profile termed star phylogeny, which suggeststhat the various HIV-1 subtypes might have evolved and divergedsynchronously from a common ancestor.

[0013] Recently, two separate viruses of this group of HIV-1 viruseswere isolated and characterized. These two viruses were obtained frompatients living in Cameroon, in West Central Africa (Gürtler, et al.1994, Vanden Heasevelde, et al. 1994). Their sequence, more particularlythe sequence of their env (envelope) gene, shows clearly that theseviruses belong to a separate category of HIV-1-related viruses, referredto as HIV-1 group O (Nkengasong et al., 1993).

[0014] However, the diversity of the isolates within this group ofHIV-1-related viruses is not known, and its propagation outside Africahas not been documented.

[0015] A general constraint, in the development of HIV serologicaltests, is to avoid both falsely positive—or falsely negative—resultswhile at the same time retaining or improving the sensitivity in termsof detection of seropositivity which the previous tests allow.

[0016] Tests based on the use of consensus peptide(s), essentiallyderived from the “env” gene, were considered as an almost ideal solutionuntil the discovery of the HIV-1-O variant brought to light thepossibility of falsely negative results (Genomic cloning and completesequence analysis of a highly divergent African human immunodeficiencyvirus isolate. J. Virol. 1994; 68: 1586-96; a new subtype of humanimmunodeficiency virus type 1 (MPV-5180) from Cameroon. J. Virol. 1994;68: 1581-85).

[0017] The non-reactivity of certain tests with “env” peptide antigen,in patients nonetheless exhibiting certain clinical syndromescharacteristic of AIDS or lymphadenopathy syndromes which occasionallyprecede them, is, at the present time, occasionally attributed to aninfection of the HIV-1-O group (HIV-1/HIV-2 seronegativity in HIV-1subtype O infected patients, Lancet 1994; 343: 1393-94; New HIV-1subtype in Switzerland. Lancet 1994; 344: 270-71).

DESCRIPTION OF THE INVENTION

[0018] The aim of the present invention is to provide diagnosticlaboratories with means, in particular specific peptides, allowing adetection of anti-HIV antibodies which were hitherto liable to beundetected. The invention also relates to mixtures of peptides obtainedfrom HIV-1 DUR and of corresponding peptides from other HIVs, so as toavoid potential “false negative” results.

[0019] The invention moreover relates to a process of detection and ofdiscrimination, in a biological sample, between antibodiescharacteristic of an HIV-1-M type retrovirus and antibodiescharacteristic of an HIV-1 group (or subgroup) O retrovirus.

[0020] The invention stems from observations made on a seropositivewoman who had stayed in the Cameroon and who had revealed an atypicalserological reactivity, in the course of several tests of screening forHIV infection, these tests having been confirmed by “Western blot”techniques.

[0021] On account of this atypical serological reactivity, in particularthe lack of reactivity to certain third-generation tests, even modifiedfor the O type, the inventors considered it interesting to carry outsequencing of certain parts of the genome of this HIV-1 DUR strain, morespecifically of the GAG and ENV genes.

[0022] However, gene amplifications by PCR using primers obtained fromthe M group and known primers from the O group were unsuccessful for theparts coding for the V3 loop of gp120, and for the immunodominant regionof gp41. Only the GAG region could be amplified using primers known inthe prior art (Loussert-Ajaka I, Lancet 1994; 343: 1393). Another aim ofthe present invention is consequently to determine primers capable ofovercoming this problem.

[0023] Partial sequences of the glycoproteins gp41 and gp120 weredetermined, along with capsid proteins (GAG gene), from lymphocyte DNAand from viral cultures, indicating that this HIV-1 DUR strain belongsin part to the HIV-1-O group, and that it differs considerably from theM group, more particularly as regards gp41 and gp120.

[0024] Thus, it was possible to demonstrate, more particularly asregards the GAG sequence of HIV-1_((DUR)), the existence of consensussequences in the O group, in several regions, which are distinct fromthe consensus sequences of the M group in the same regions.

[0025] Cloning of the sequences coding for the GAG, gp41 and gp120fragments of HIV-1_((DUR)) was carried out in a Bluescript® plasmidcontaining a PST1 site. The amplification products were cloned eitheraccording to the standard techniques using T3 and T7 universal primers,or directly sequenced by using the primers of the precedingamplification. The sequences were then determined with the AppliedBiosystems 373A automated sequencer (ESGS Montigny le Bretonneux,France).

[0026] Within the context of the present invention, the inventors haveisolated and sequenced the env gene from an O group isolate,HIV-1_((VAU)), obtained from a French patient who had never traveledoutside Europe and who died of AIDS in 1992. According to its envelopesequence, HIV-1_((VAU)) is related to two recently characterizedCameroonian viruses HIV_(ANT70) and HIV_(MVP5180). Phylogenetic analysisof the env sequences reveals that the three viruses appear to constitutea separate group, which will be referred to herein as HIV-1 group O. Theisolation of HIV-1_((VAU)) from this patient also indicates that adegree of propagation of HIV-1 group O has already occurred outsideAfrica.

[0027] Isolation of the HIV-1_((VAU)) virus

[0028] HIV-1_((VAU)) was isolated in 1992 from a 41-year-old Frenchpatient suffering from AIDS. This patient exhibited, in 1986, a severeleuconeutropenia associated with a carcinoma of the uterine cervix.However, she gradually showed signs of opportunistic infections, with areduction in the number of circulating CD4⁺ T cells and she died of AIDSin 1992. Anti-HIV-1 antibodies were first detected by ELISA (Elavia,Sanofi Diagnostics Pasteur and Abbott test) in 1990.

[0029] The patient had never traveled outside Europe, had not usedintravenous medicinal products and had not received any known bloodtransfusion. No sexual partner of African origin has been identified.She gave birth to a healthy child in 1971, but a son, born in 1980, diedat the age of one following a clinical episode highly suggestive ofneonatal AIDS. Her third child, born in 1983, and her husband arecurrently in good health and not infected.

[0030] The isolation of the virus was carried out in the followingmanner: the CD8⁺ cells present in the PBMCs (peripheral bloodlymphocytes) of the patient were removed using beads coated with IOT8antibodies (Immunotech). These remaining PBMCs were stimulated with PHA,then cocultured with CD8-depleted PBMCs obtained from a healthy donorand stimulated with PHA. Viral growth in the coculture was monitored byassaying the reverse transcriptase (RT) activity of the supernatant andby ELISA test of the HIV-1 p24 (diagnostic kit marketed by DuPont deNemours). The virus obtained from the initial coculture was subjected toseveral passages in CD8-depleted and PHA-stimulated PBMC cultures.Several attempts were made to infect, with the HIV-1_((VAU)), varioustransformed cell lines, including MT4 cells (Harada, et al. 1985) andCEM cells (Rey, et al. 1989), as well as the Hela-CD4-LTRLacZ cell lineP4-2 (Clavel and Charneau 1994).

[0031] Biological Characterization of HIV-1_((VAU))

[0032] Two weeks after coculturing the patient's CD8-depleted,PHA-stimulated PBMCs with similar cells from a healthy donor, theproduction of virus was detected in the form of an RT activity peak inthe culture supernatant. This virus could then be subjected to serialpassages on CD8-depleted, PHA-stimulated normal PBMCs. In FIG. 1, plateA represents the production of HIV-1_((VAU)) in infected PBMC culturesupernatants, checked by RT assay (filled circles) and HIV-1 p24 antigencapture ELISA (empty circles). The concentration of HIV-1 p24 isexpressed in ng/ml and the RT activity in cpm/μl. In plate B, the sameexperiment was carried out with a standard primary HIV-1 isolate from anAIDS patient.

[0033] Although the growth of HIV-1_((VAU)) was easily detected by RTassay, the detection of virus in the culture supernatants by HIV-1 p24ELISA (DuPont) was substantially less sensitive. FIG. 1 shows thecomparison between the profiles of productive infection of PBMCs eitherwith HIV-1_((VAU)) or with a primary HIV-1 isolate from an AIDS patient,assayed by RT or p24. For equivalent quantities of particles, determinedby assay of RT activity in the supernatants assayed, approximately 25times less p24 was detected in the case of HIV-1_((VAU)) than in thecase of the other HIV-1 isolate. The difference may be due to the factthat the monoclonal antibody specific for HIV-1 p24, which is used tocoat the ELISA plates, has only a weak affinity for the gag products ofHIV-1_((VAU)).

[0034] Several negative attempts were made to propagate HIV-1_((VAU)) ontransformed human T cell lines sensitive to HIV-1. In particular,cocultures between PBMCs infected with HIV-1(VAU) and either MT4 cellsor CEM cells did not lead to propagation of the virus. It was also foundthat this virus was not capable of infecting CD4⁺ HeLa cells (P4-2)(Clavel and Charneau 1994) carrying a lacZ gene inducible by the tatgene. Likewise, no replication of HIV-1_((VAU)) could be detected inactivated peripheral blood lymphocytes from several chimpanzees.

[0035] Analysis of the HIV-1_((VAU)) envelope sequence, which will bedescribed in detail later, and its comparison with that of the tworecently described Cameroonian isolates indicate that all three virusesbelong to the same group of HIV-1-related viruses. Furthermore, thiscomparison indicates that these three variants of the virus areapproximately phylogenetically equidistant from each other.Consequently, each of the three virus variants constitutes on its own adistinct subtype of their group, which is now called HIV-1 group O. Thisgroup is different from the group of other HIV-1 isolates, identified upuntil now, which the inventors call here HIV-1, group M.

[0036] The appearance of this new group poses the question of itsorigin: did group O evolve from group M viruses (or conversely) or doeseach group have a different history? The inventors think that, insofaras both group M and group O have a similar internal divergence profile,it is likely that they each correspond to the diversification ofdistinct viral ancestors in distinct human populations. It is notpossible to assess from the phylogenetic and virological data currentlyavailable whether the ancestor of either of the two groups affectedhumans naturally or was introduced into humans from other species. Theonly virus similar to HIV-1 present in a nonhuman primate is theSIVCPZGAB isolate (Huet, et al. 1990), isolated from a chimpanzeeapparently infected naturally, which is clearly different both fromgroup M and from group O, and for which no human equivalent has beenfound. It is unlikely that the group O viruses evolved recently from achimpanzee virus insofar as HIV-1_((VAU)) has not succeeded inreplicating in chimpanzee lymphocytes.

[0037] Why does the group O epidemic appear only now, some 15 to 20years later than group M? There are three possible explanations:firstly, the introduction of the ancestor of the group O viruses intohumans is thought to have occurred more recently than that of group M;secondly, it is possible that group M was allowed to spread earliercompared with group O because of different social conditions in theirregion of origin; and thirdly, the group O viruses could have a lowercapacity For transmission compared with that of the group M viruses. Ithas been proposed that such a property explains the absence ofsignificant worldwide propagation of HIV-2, for which a smaller viralload in infected subjects is linked to reduced transmissibility (DeCock, et al. 1993). In this regard, although no data are available onthe viral load in patients infected with an HIV-1 group O, thepathogenicity of these viruses does not appear to be different from thatof HIV-1. The patient from whom HIV-1_((VAU)) was isolated died of AIDS,like the patient from whom the HIV_(MVP5180) group O isolate wasobtained.

[0038] However, the natural history of infection of the HIV-1_((VAU))patient is still not clear, but there are several indications that thispatient was infected before 1980, as suggested by the death on that dateof her second child suffering from a syndrome resembling AIDS.

[0039] The invention relates to any variant of the nucleic acidsequences of the HIV-1_((VAU)) virus or of any group O equivalent virus,containing structural proteins which have the same immunologicalproperties as the structural proteins coded for by the env genecomprising the sequence described in FIG. 6 and called “vau”, alsodesignated by SEQ ID No. 5.

[0040] The present invention also relates to compositions containingeither antigens according to the invention, or a mixture of antigensaccording to the invention combined with extracts originating from oneor more HIV-1 group O viruses or from other variant viruses, on the onehand, and from one or more HIV-2 and/or HIV-1 viruses, on the otherhand, these compositions being optionally labeled. It is possible to useany type of appropriate label: enzymic, fluorescent, radioactive, etc.

[0041] Nucleic Acids

[0042] The invention relates to the DNAs or DNA fragments, moreparticularly cloned DNAs and DNA fragments, obtained from RNA, cDNA orprimers which can be used in PCR, or other gene amplification methods,derived from the HIV-1_((VAU)) retrovirus RNA or DNA. The inventionrelates more particularly to all the equivalent DNAs, especially to anyDNA having sequence homologies with the HIV-1_((VAU)) DNA, in particularwith the sequence coding for for the env region of the HIV-1_((VAU))strain comprising the sequence corresponding to SEQ ID No. 5 representedin FIG. 6 and called “vau”. The homology with HIV-1 group M is at leastequal to 50%, preferably to 70% and still more advantageously to about90%. Generally, the invention relates to any equivalent DNA (or RNA)capable of hybridizing with the DNA or RNA of a group O HIV-1retrovirus.

[0043] The invention also relates to the RNA sequences corresponding tothe DNA sequences defined above.

[0044] The invention also relates to the HIV-1_((VAU)) virus integrasegene comprising the sequence identified by the name SEQ ID No. 7 orhybridizing with SEQ ID No. 7. The invention also relates to the RNAscorresponding to the DNA described above.

[0045] The subject of the invention is also compositions containing thepeptides or polypeptides encoded by the abovementioned DNA or DNAfragments.

[0046] Oligonucleotides derived from the VAU sequence or alternativelyfrom the HIV-1_((VAU)) virus integrase gene, particularlyoligonucleotides comprising at least 9 nucleotides, may be used for thedetection of group O HIV-1 virus DNA or RNA sequences in biologicalsamples, cell cultures or cell extracts, by the PCR technique or anyother gene amplification technique. These sequences could be used eitheras gene amplification primers or as probes for the specific detection ofthe gene amplification products. Also capable of being used ashybridization probes are the amplification products, or theircorresponding synthetic sequence, obtained by chemical synthesis(Applied Biosystems).

[0047] The invention also covers any fragment of at least 100nucleotides which may be used as a probe in hybridization reactions andcapable of permitting reaction with part of the genome of anHIV-1_((VAU)) variant under high stringency hybridization conditions.

[0048] Cloning and Sequencing of the HIV-1_((VAU)) env Gene

[0049] For the initial PCR amplification of the HIV-1_((VAU)) DNA, thetotal DNA was extracted from PBMCs infected with HIV-1_((VAU)) and asegment of the pol gene (integrase region) was amplified usingdegenerate primers:

[0050] primer 4506: 5′AGTGGAT(A/T) (T/C)ATAGAAGCAGAAGT3′; Seq. ID No. 1;

[0051] primer 5011: 5′ACTGC(C/T)CCTTC(A/C/T)CCTTTCCA3′; Seq. ID No. 2;

[0052] The reaction medium including 50 mM KCl, 10 mM Tris-HCl (pH 8.9),1.5 mM MgCl₂, 0.1 mg/ml gelatin, 0.2 mM DNTP, 1U of Taq polymerase(Amersham). The PCR was carried out in 43 thermal cycles at 92° C. for10 seconds, 50° C. for 1 minute and 72° C. for 40 seconds.

[0053] The resulting amplification product was cloned into a pBluescriptvector, generating the clone ph4, deposited at the CNCM on Oct. 20, 1994under No. I-1486, which was subsequently used as a probe to screen alambda library of low molecular weight DNA, which was digested withEcoRI and was obtained from cells infected with HIV-1_((VAU)). Briefly,the PBMCs infected with HIV-1_((VAU)) were cocultured for 24 hours withnew PBMCs stimulated with PHA and depleted of CD8⁺ cells, after which ahigh cytopathic effect (CPE) was visible. The low molecular weight DNAwas then extracted according to the Hirt method (Hirt 1967), anddigested with the enzyme EcoRI. A previous Southern-blot analysis ofthis DNA had indeed shown that the HIV-1_((VAU)) genome contained onlyone EcoRI site, permitting the cloning of nonintegrated circular DNAspecies representing the entire viral genome. The resulting digestionproduct was subjected to agarose gel electrophoresis, and the populationof DNA fragments of approximately 8-12 kb in size was purified andligated to EcoRI-digested lambda Zap DNA (Stratagene). Afterencapsidation, plating and screening by hybridization with ³²P-labeledph4 DNA, a clone, λH34, was identified as being positive, and amplified.The EcoRI insert was purified, sonicated, and cloned by the “shotgun”technique into the phosphatase-treated vector M13mp18 digested with theenzyme SmaI. One hundred and fifty of the clones obtained were sequencedin a 373A DNA sequencer (Applied Biosystems), and the resultingsequences were assembled into a single sequence using the Wisconsin GCGDNA analysis package.

[0054] Analysis of this sequence revealed numerous nonsense codons inall the reading frames, which is highly suggestive of a hypermutatedgenome (Vartanian, et al. 1991). This sequence being unusable, it wasconsequently decided to amplify, by PCR, the HIV-1_((VAU)) env geneusing the total DNA from PBMCs infected with HIV-1_((VAU)), and theoligonucleotide primers derived from the sequence λH34:

[0055] primer TH2 5′GCTCTAGATGGGGATCTCCCATGGCAGG3′ Seq. ID No. 3;

[0056] primer UH2 5′GCTCTAGATCAGGGAAGAATCCCTGAGTGT3′. Seq. ID No. 4.

[0057] The PCR amplification was carried out in 35 thermal cycles at 92°C. for 15 seconds, 52° C. for 1 minute, 60° C. for 2 minutes and 72° C.for 2 minutes. The resulting amplification product, of 3.5 kb in size,was cloned into the M13mp18 vector and sequenced by successivereactions, first using the M13 universal sequencing primer, and then theprimers deduced from the upstream sequences. Analysis of the nucleotideand peptide sequences was carried out using the Wisconsin GCG DNAanalysis package. The HIV-1_((VAU)) env gene codes for 877 amino acidsin total, including the signal peptide. The nucleotide sequence of theHIV-1_((VAU)) env gene corresponds to Seq. ID No. 5 (see FIG. 3).

[0058] Use of Nucleic Acids as Probes

[0059] The invention also relates naturally to the use of DNA, cDNA orfragments thereof, or of recombinant plasmids or other equivalentvectors containing these fragments, as probes, for detecting thepresence or otherwise of the HIV-1_((VAU)) virus in serum samples orother biological fluids or tissues obtained from patients suspected ofbeing carriers of the HIV-1_((VAU)) virus. These probes are optionallylabeled (radioactive, enzymic or fluorescent labels and the like).Probes which are particularly valuable For the implementation of themethod for detecting the HIV-1_((VAU)) virus or an HIV-1_((VAU)) variantmay be characterized in that they comprise all or a fraction of the DNAcomplementary to the HIV-1_((VAU)) virus genome or alternativelyespecially the fragments contained in various clones. An HIV-1_((VAU))cDNA fraction containing all or part of the env region will be mentionedmore particularly.

[0060] The probes used in this method for detecting the HIV-1_((VAU))virus or in diagnostic kits are not in any way limited to the probesdescribed previously. They comprise all the nucleotide sequencesobtained from the genome of the HIV-1_((VAU)) virus, an HIV-1_((VAU))variant or a virus similar by its structure, provided that they allowthe detection, using biological fluids from individuals likely to haveAIDS, of an HIV-1 group O virus, in particular HIV-1_((VAU)) byhybridization with the HIV-1_((VAU)) virus DNA or RNA.

[0061] Particularly advantageous are the probes which, when hybridizedwith HIV-1, give a strong reaction with HIVs belonging to group O and aweak reaction with HIVs belonging to group M. By way of nonlimitingexample, a probe constructed from the HIV-1_((VAU)) virus integrase genesequence (SEQ ID No. 7) gives, when it is hybridized with HIV-1 underhybridization conditions such as those described in Patent EP 178 978, astrong reaction with group O HIVs and a weak reaction with group M HIVs.

[0062] The detection may be performed in any manner known per se,especially:

[0063] by bringing these probes into contact either with the nucleicacids obtained from cells contained in biological fluids (for examplespinal fluid, saliva and the like), or with these fluids themselves,provided that their nucleic acids have been made accessible tohybridization with these probes, and this under conditions permittinghybridization between these probes and these nucleic acids,

[0064] and by detecting the hybridization which may be produced.

[0065] The abovementioned diagnosis involving hybridization reactionsmay also be performed using mixtures of probes derived fromHIV-1_((VAU)), HIV-1 and HIV-2 respectively, provided that it is notnecessary to make a distinction between the desired HIV virus types.

[0066] The subject of the invention is also expression vectorscontaining the sequence coding for the HIV-1 envelope proteins orcontaining the sequence coding for the integrase.

[0067] The invention comprises compositions for detecting the presenceor otherwise of the HIV-1_(VAU) virus in serum samples or samples ofother biological fluids or tissues, obtained from patients likely to becarriers of the HIV-1_(VAU) virus. These compositions are characterizedin that they comprise at least one probe obtained from a nucleotidesequence obtained or derived from the HIV-1_(VAU) virus genome,particularly an HIV-1_(VAU) DNA fragment containing the region or partof the region coding for for the env protein of the HIV-1_(VAU) virus orof an HIV-1_(VAU) variant.

[0068] Advantageously, the composition described above also comprises aprobe obtained from a nucleotide sequence derived from HIV-1 or HIV-2.

[0069] Other diagnostic compositions comprise the primers of theinvention which are capable of being used in gene amplification ofsubgroup O retroviruses or variants of these retroviruses.

[0070] Antigens, Especially Proteins and Glycoproteins

[0071] The invention relates to an HIV-1 group (or subgroup) Oretroviral protein, or natural or synthetic peptide or polypeptidecomprising at least a part of said protein, which is capable of beingrecognized by antibodies which may be isolated from serum obtained afteran infection with an HIV-1 group O VAU strain, or an HIV-1 group O DURstrain.

[0072] The invention relates to an external envelope protein of theHIV-1_(VAU) retrovirus encoded by the gene comprising the sequencecorresponding to SEQ ID No. 5. According to a preferred embodiment ofthe invention, this protein is in addition characterized in that itcomprises the amino acid sequence corresponding to Seq ID No. 6represented in FIG. 3 and comprising amino acid residues 1 to 526. Thesubject of the invention is also any polypeptide or variant which isderived from said sequence having an epitope which may be recognized byantibodies induced by the HIV-1_(VAU) virus.

[0073] The abovementioned protein may be obtained in a glycosylated ornonglycosylated form.

[0074] The subject of the invention is also an envelope transmembraneprotein comprising the amino acid sequence SEQ ID No. 8 represented inFIG. 3 between amino acid residues 527 and 877. This transmembraneprotein is, within the scope of the invention, in glycosylated ornonglycoslated form.

[0075] The invention relates to all the antigens, especially proteins,glycoproteins, polypeptides or peptides, obtained by expressing codingsequences of the HIV-1_((VAU)) genome and having immunologicalproperties equivalent to those of HIV-1_((VAU)). The antigens are saidto be equivalent within the scope of the present invention provided thatthey are recognizable by the same antibodies, especially antibodieswhich can be isolated from serum obtained from a patient who has beeninfected with an HIV-1_((VAU)).

[0076] In particular, the subject of the invention is the peptides orpolypeptides which are synthesized chemically and whose amino acidsequence is contained in that of the HIV-1_((VAU)) envelope proteins,which sequence is represented in FIG. 3, or the equivalent peptides orpolypeptides.

[0077] There should also be included among the equivalent peptides,polypeptides, proteins or glycoproteins, the fragments of the aboveantigens and the peptides which are prepared by chemical synthesis or bygenetic engineering, so long as they give rise to immunologicalcross-reactions with the antigens from which they are derived. In otherwords, the invention relates to any peptide or polypeptide havingepitopes which are identical or similar to the epitopes of theabovementioned antigens and which are capable of being recognized by thesame antibodies. Forming part of this latter type of polypeptides arethe products of expression of DNA sequences corresponding to DNAsequences coding for for the polypeptides or antigens mentioned above.

[0078] More particularly, the antigens which are obtained from theHIV-1_((VAU)) virus or produced by genetic engineering or conventionalchemical synthesis, and which are of the greatest interest within thecontext of the present invention, are the antigens which make itpossible to obtain a clear distinction between the HIV-1_((VAU)) virusesof the invention and the viruses of the HIV-1 and HIV-2 groups. In thisregard, considerable differences have been observed at the level of theHIV-1_((VAU)) virus envelope protein as well as at the level of theimmunodominant epitope of the external portion of the PM protein. Itappears that the gag and pol proteins exhibit greater similarity withthe HIV-1 virus than the envelope protein.

[0079] The invention also relates to peptides or polypeptides which areidentical to the immunodominant region of the HIV-1_((VAU)) envelopetransmembrane glycoprotein. This region is represented in FIG. 3.

[0080] Preferred polypeptides of this region are, for example, thosewhich contain the sequence CKNRLIC or correspond to this sequence. Theymay also be peptides or polypeptides corresponding to the sequenceRLLALETFIQNWWLLNLWGCKNRLIC or comprising this sequence.

[0081] Another preferred peptide, identified below by the name “VAUpeptide”, corresponds to the following sequence or comprises thissequence or any part of this sequence capable of being recognized byantibodies directed against the HIV-1_((VAU)) retrovirusRARLLALETFIQNQQLLNLWGCKNRLICYTSVKWNKT.

[0082] Variant polypeptides of this sequence are for example thepolypeptides represented in FIG. 4 for the HIV-1_((MVP5180)) andHIV-1_((ant70)) isolates. These polypeptides may also be derived fromthe preceding ones by insertion and/or deletion and/or substitution, forexample conservative substitution by amino acid residues.

[0083] The present invention relates to a peptide obtained from theHIV-1-O DUR virus deposited on Feb. 23, 1995 at the CNCM under thereference I-1542, or a peptide whose sequence is distinguished from thatof the above by substitution, deletion or addition of amino acids, thisseparate peptide nevertheless retaining the antigenic characteristics ofthe above one.

[0084] Other peptides falling within the scope of the invention aredefined below.

[0085] Thus, a preferred peptide of the invention is a peptidecontaining at least 4 consecutive amino acids contained in the GAGsequence represented in FIG. 8 or in an immunologically similar GAGsequence obtained from a variant of the HIV-1-O DUR virus, saidimmunologically similar sequence being recognized by antibodies whichalso specifically recognize at least one of the sequences AHPQQA,LWTTRAGNP contained in the GAG sequence of FIG. 8.

[0086] Preferably, this peptide consists of a peptide whose amino acidsequence is contained either in one of the following sequences:SPRTLNAWVKAVEEKAFNPEIIPMFMALSEGA (1) MLNAIGGHQGALQVLKEVIN (2)GPLPPGQIREPTGSDIAGTTSTQQEQI (3) IPVGDIYRKWIVLGLNKMVKMYSPVSILDI (4)QGPKEPFRDYVDRFYKTKLAE (5) AHPQQA (5a) LWTTRAGNP (5b)

[0087] or in a corresponding immunologically similar sequence, thispeptide containing at least 4 consecutive amino acids of one of saidsequences.

[0088] Preferably also, this peptide consists of a peptide whose aminoacid sequence is contained either in one of the following sequences:SPRTLNAWVK (6) GSDIAGTTST (7) QGPKEPFRDYVDRF (8)

[0089] or in a corresponding immunologically similar sequence, thispeptide containing at least four consecutive amino acids of one of saidsequences.

[0090] Peptides which are particularly preferred in the presentinvention are the peptides containing:

[0091] the amino acid sequence NPEI (9) or

[0092] the amino acid sequence AVEEKAFNPEIIPMFM (10), and moreparticularly peptides whose amino acid sequence is contained, either inone of the following sequences: IGGHQGALQ (23) REPTGSDI (24)

[0093] or in a corresponding immunologically similar sequence, thispeptide containing at least 4 consecutive amino acids of one of saidsequences, as well as the peptide whose amino acid sequence iscontained, in the following amino acid sequence:

[0094] INDEAADWD (25)

[0095] or in a corresponding immunologically similar sequence, thispeptide containing at least 4 consecutive amino acids of said sequence.

[0096] The present invention relates to the nucleic acid sequencescoding for peptides (23), (24) and (25) as well as the nucleic acidsequences coding for the immunologically similar sequences, as well ascompositions comprising at least one of these nucleic acids.

[0097] The invention also relates to the use of at least one of thesenucleic acids for detection and discrimination between HIV-1 group M andHIV-1 group O strains.

[0098] A peptide derived from the RIV-1-O DUR virus defined above alsofalls within the scope of the present invention, said peptide containingat least 4 consecutive amino acids of the V3 loop of gp120 representedin FIG. 9 or of the corresponding immunologically similar sequence,obtained from a variant of the HIV-1-O DUR virus, said immunologicallysimilar sequence being recognized by antibodies which also specificallyrecognize at least one of the sequences: KEIKI (12), EREGKGAN (13),CVRPGNNSVKEIKI (14), QIEREGKGANSR (15).

[0099] This peptide preferably contains:

[0100] a) either the sequence CVRPGNNSVKEIKIGPMAWYSMQIEREGKGANSRTAFC(11) or a part of this sequence which contains at least 4 amino acids

[0101] b) or an amino acid sequence which is separate from the sequenceof a) in which one or more amino acids are replaced with one or moreamino acids, with the proviso that the peptide retains its reactivitywith an antiserum against the abovementioned peptide,

[0102] c) or an amino acid sequence which is separate from a) or b), inwhich one or more amino acids have been deleted or added, with theproviso that the peptide retains its reactivity with an antiserumagainst the peptide of a),

[0103] d) or a corresponding immunologically similar sequence or part ofsequence. Preferably also, this peptide contains either the sequenceKEIKI (12), or the sequence EREGKGAN (13), or the sequence GPMAWYSM(16).

[0104] In a particularly preferred manner, a peptide as defined abovecontains the amino acid sequence CVRPGNNSVKEIKI (14) or the sequenceQIEREGKGANSR (15).

[0105] A peptide derived from the HIV-1-O DUR virus as defined abovealso falls within the scope of the invention, said peptide containing atleast 4 consecutive amino acids, whose entire sequence is contained inthe sequence of the immunodominant region of gp41 represented in FIG. 9or in a corresponding immunologically similar sequence, obtained from avariant of the HIV-1-O DUR virus, said immunologically similar sequencebeing recognized by antibodies which also specifically recognize atleast one of the following sequences:

[0106] RLLALETLMQNQQL (17), LNLWGCRGKAICYTSVQWNETWG (18), CRGKAI (19),SVQWN (20), RLLALETLMONQQLLNLWGCRGKAICYTS (21), QNQQLLNLWGCRGKAICYTSVQWN(22).

[0107] This peptide is preferably a peptide containing the sequenceRLLALETLMQNQQL (17), or LNLWGCRGKAICYTSVQWNETWG (18) or part of thispeptide (18) containing:

[0108] a) either the sequence CRGKAI (19) or the sequence SVQWN (20) inwhich Q is, where appropriate, replaced by a different amino acid, whichis nevertheless also different from K, or the two sequences at the sametime,

[0109] b) or an amino acid sequence which is separate from the sequenceof a) in which one or more amino acids are replaced with two aminoacids, with the proviso that the peptide retains its reactivity with anantiserum against the peptide of a),

[0110] c) or an amino acid sequence which is separate from a) or b), inwhich one or more amino acids have been deleted or added, with theproviso that the peptide retains its reactivity with an antiserumagainst the peptide of a),

[0111] d) or in a corresponding immunologically similar sequence or partof sequence.

[0112] Preferably also, this peptide possesses one or the other of thefollowing characteristics:

[0113] its N-terminal sequence which contains at least 8 amino acids isnot immunologically recognized by antibodies formed against the sequenceRILAVERY contained in the immunodominant region of gp41 of the HIV-1-LAIstrain.

[0114] it is not recognized by antibodies formed against the peptideSGKLIC of the HIV-1-LAI strain.

[0115] it contains either of the following two sequences:RLLALETMONQQLLNLWGCRGKAICYTS (21) QNQQLLNLWGCRGKAICYTSVQWN (22)

[0116] Synthesis of VAU Peptides

[0117] A VAU peptide was prepared by the conventional solid phasepeptide synthesis technique using the “continuous flow” Fmoc method. Thepeptide was prepared using a Milligen 9050 PEP synthesizer and using the“Millipore” PEG PAL resin, substituted with the first C-terminal aminoacid residue. The side chains of the amino acids are protected by thefollowing groups: Pmc for arginine; Trt for asparagine, glutamine andcysteine; Boc for lysine; tBu ester for glutamic acid; tBu ether forserine, threonine and tyrosine. The temporary Fmoc groups are removedwith a 20% piperidine solution in DMF. The reactions for coupling eachamino acid are performed with 6 equivalents of DIPCDI and HOBT. Someresidues require a double coupling especially arginines 1 and 23,cysteines 19 and 26, asparagine 11, glutamines 10, 12 and 13, alanine 4,isoleucine 9 and leucines 2, 3, 14 and 15.

[0118] After coupling, the resin is dried under vacuum. The peptide iscleaved from the support by the K reagent for 4 hours at roomtemperature. The crude peptide is precipitated and washed with ethylether. The product is purified by high-pressure liquid chromatography(HPLC) in a WATERS LC PREP 4000 instrument with WATERS Delta Pak C1840×100 mm cartridges, flow rate 30 ml/min, acetonitrile/0.1% TFAgradient. The fractions containing the peptide are combined,concentrated in a rotary evaporator and then lyophilized.

[0119] Cyclization

[0120] The peptide (0.025 mM) is dissolved in a 10 mM ammonium acetatesolution. The pH is adjusted to 8.5 with 1M ammonium hydroxide solution.The pH is readjusted after 3 or 4 hours. The cyclization is monitored byHPLC at 214 nm and 280 nm, WATERS Delta Pak C18 5μ column,acetonitrile/0.1% TFA gradient. The cyclization is complete after 15hours. The pH is brought to 6 using 97-100% acetic acid, the solution islyophilized and then purified under the same conditions as for the crudepeptide.

[0121] The peptide is checked by HPLC and by mass spectrometry accordingto the electrospray technique (FISON VG Trio 2000 spectrophotometer).Fmoc: 9-Fluoroenylmethyloxycarbonyl Pmc:8-Methylpentane-6-sulfonylchroman Trt: Tritryl Boc: TertbutyloxycarbonyltBU: tert butyl DMF: Dimethylformamide DIPCDI: DiisopropylcarbodiimideHOBT: 1-Hydroxybenzotriazole TFA: Trifluoroacetic acid Reagent K:Phenol/water/thioanisole/ethanedithiol/TFA; 2.5 ml/2.5 ml/2.5 ml/1.5ml/41 ml

[0122] Comparison of the amino acid sequence of the HIV-1_((VAU))envelope with the corresponding sequence of other HIV viruses.

[0123] Western-blot analysis of a series of serum samples obtained froma patient infected with HIV-1_((VAU)) is presented in FIG. 2.Nitrocellulose strips, carrying proteins separated by electrophoresisand obtained from purified HIV particles (LAV BLOT, SANOFI DIAGNOSTICSPASTEUR), were incubated with serum samples and their reactivity wasevaluated according to the procedures recommended by the manufacturer.The results obtained are the following: strip 1: proteins specific forHIV-2, which have been reacted with a serum sample obtained in February1992 from the HIV-1_((VAU)) patient. Strips 2-7:HIV-1 positive sera;sera from the HIV-1_((VAU)) patient: 2: obtained in November 1990; 3: inDecember 1990; 4: in February 1991; 5: in February 1992; 6: negativecontrol; 7: positive control (serum from an individual infected withHIV-1). The names and the size of the proteins (in kD) are indicated inthe margin.

[0124]FIG. 3 shows an alignment of the amino acid sequence of theHIV-1_((VAU)) envelope with the corresponding sequence of the HIV-1-LAIreference isolate (Wain-Hobson, et al. 1985). The signal peptides, theV3 loop and the gp41 immunodominant epitope are highlighted by shadedrectangles. The site of cleavage between the external envelopeglycoprotein gp120 and the transmembrane gp41 is indicated by arrows.The vertical lines between the amino acid letters indicate completeidentity, colons (:) indicate high homology, and dots (.) indicatelimited homology between individual amino acids. The alignment wasperformed using the GAP program of the Wisconsin GCG package.

[0125] The original version (1.0) of the GAP and BESTFIT programs waswritten by Paul Haeberli from a detailed study of the publications ofNeedleman and Vunsch (J. Mol. biol. 48, 443-453 (1970) and of Smith andWaterman (Adv. Appl. Math. 2; 482-489 (1981). The limited alignmentswere developed by Paul Haeberli and added to the package to constitutethe 3.0 version. They were then fused into a single program by PhilipMarquess to constitute the 4.0 version. The gap absence penalties in thealignment of the proteins were modified as suggested by Rechid, Vingronand Argos (CABIOS 5; 107-113 (1989)).

[0126] The alignment of FIG. 3 shows numerous regions of highdivergence, with a few domains retained here and there. These retainedregions correspond roughly to the domains also retained in theconventional HIV-1 isolates (Alizon et al. 1986, Benn et al. 1985).Among the divergent domains, the V3 loop, also called principaldeterminant of neutralization (Javaherian et al. 1990, Javaherian et al.1989, Matsushita et al. 1988) is clearly one of the most divergent,although the two cysteines defining the loop are retained. The sequenceof the cap of the loop, GPGRAF for HIV-1-LAI is GPMAWY in HIV-1_((VAU)).This unit of the cap is identical to that of the Cameroonian group Oisolate HIV_((ANT70)) (Van den Heasevelde et al. 1994), but is differentfrom that of the other group O isolate, HIV_(MVP5180) (Güartler et al.1994), for which the motif is GPMRWR.

[0127] In the entire envelope, 29 potential N-glycosylation sites wereidentified in total, of which 13 are retained compared with other HIV-1envelope proteins. 19 retained cysteines were also found in total, whichindicates that the overall folding architecture of the protein isretained, but 5 nonretained cysteines were found.

[0128]FIG. 4 shows the multiple alignment of the immunodominant peptidesin the extracellular segment of the transmembrane envelope glycoproteinof various HIV-1 isolates. All the sequences are compared to theHIV-1-LAI reference sequence. Hyphens indicate identity with HIV-1-LAI.The alignment was made with the aid of the PILEUP program of theWisconsin GCG package.

[0129] In the PILEVP program, the assembling strategy represented by thedendrogram is called UPGMA, which means “unweighted pair-group methodusing arithmetic averages” (Smith, P. H. A. Sokal, R. R. (1973) inNumerical Taxonomy (pp. 230-234), W. H. Freeman and Company, SanFrancisco, Calif., USA). Each pair alignment in PILEVP uses theNeedleman and Wunsch method (journal of Molecular Biology 48; 443-453(1970)).

[0130] As shown in FIG. 4, the amino acid sequence of the immunodominantepitope of the external portion of the TM protein (Gnann et al., 1987)is substantially different from that of other HIV-1 and HIV-2 isolates.However, it retained most of the amino acids which were found to beconserved between the HIV-1 and HIV-2 viruses.

[0131] It was found that some specific amino acids were conserved onlybetween group O viruses: such is the case for lysine in position 21 in apeptide of 26 amino acids, for threonine in position 7 and asparagine inposition 11. These differences could explain the absence of detection ofconventional HIV-1 envelope antigens by one of the sera from theHIV-1_((VAU)) patient and also probably by that of the patients infectedby other group O viruses. Overall, the comparison between the HIV-1-LAIand HIV-1_((VAU)) envelope sequences showed a 50% identity. TheHIV-1_((VAU)) envelope sequence was also compared to that of other HIVrepresentatives including the two members of HIV-1 group O described andsequenced: HIV-1_(ANT70) and HIV-1_(MVP5180) and SIV representatives.The results of this analysis, which are presented in Table 1, establishthat HIV-1_((VAU)) belongs to group O. The HIV-1(VAU) envelope is 70%identical to the HIV-1_(ANT70) envelope and 71% identical toHIV-1M_(VP5180).

[0132] Among the most common HIV-1 subtypes, the identity at the levelof the envelope is comparable, ranging from 74% to 80%. HIV-1_(EU) 77HIV-1_(MAL) 76 80 HIV-1_(U455) 75 74 76 SIV_(CPZOAB) 61 62 63 63HIV-2_(ROD) 38 39 39 40 39 SIV_(MAC251) 37 37 38 38 38 74 SIV_(AOMTYO)37 36 37 38 40 46 47 HIV_(ANT70) 51 52 52 52 53 33 32 33 HIV_(MVP5180)51 52 54 51 52 36 33 34 70 HIV_(VAU) 50 51 52 51 54 35 34 35 70 71HIV-1_(LAI) HIV-1_(EU) HIV-1_(MAL) HIV-1_(U455) SIV_(CPZOAB) HIV-2_(ROD)SIV_(MAC251) SIV_(AOMTYO) HIV_(AHT70) HIV_(MPV5180)

[0133] The relationship between HIV-1_((VAU)), other members of thephylogeny of HIV-1 viruses and the two viruses of group O recentlydescribed was analyzed by constructing a phylogenetic tree of unweightedparsimony using the nucleotide sequence of the env transmembrane region.The result of this analysis is represented in FIG. 5 in which thenumbers indicate the number of nucleotide changes. FIG. 5 shows thatHIV-1_((VAU)) is roughly equidistant from the other two group O virusesand that, overall, these three viruses appear to be approximatelyequidistant from each other. Indeed, the number of nucleotide changesbetween HIV-1_(MVP5180) and HIV-1_((VAU)) is 218 in the segment of thegenome analyzed, whereas the distance is 183 between HIV-1_(MVP5180) andHIV-1_(ANT70), and 213 between HIV-1_(ANT70) and HIV-1_((VAU)). Thisdivergence profile is very similar to that which exists in all the otherHIV-1 subtypes, where the number of single nucleotide changes whichexist between two different subtypes ranges from 157 (subtype E tosubtype F) to 219 (subtype A to subtype D).

[0134] Table 1 shows the comparison of the envelope sequences ofdifferent viruses related to HIV-1. The numbers indicate the percentageof amino acid identity between the envelope sequences, as calculatedusing the GAP program of the Wisconsin GCG package. *: in the case ofHIV_(ANT70), only the external envelope protein was used in thecomparison.

[0135] Compositions Comprising HIV-1_((VAU)) antigens Generally, theinvention relates to any composition which can be used for the in vitrodetection of the presence, in a biological fluid, especially fromindividuals who have been brought into contact with HIV-1_((VAU)), orwith antibodies against at least one of the HIV-1_((VAU)) antigens. Thiscomposition can be applied to the selective diagnosis of infection by anHIV-1 group O by using diagnostic techniques such as those described inPatent Applications EP 84401,834 and EP 87400,151,4. Within the contextof the present invention, any constituent comprising antigenicdeterminants capable of being recognized by antibodies produced againstHIV-1_((VAU)) is used, for example recombinant antigens or peptides orchemically synthesized peptides defined from the sequence of theHIV-1_((VAU)) envelope. In this regard, the invention relates moreparticularly to compositions containing at least one of theHIV-1_((VAU)) virus envelope proteins. There may be mentioned, by way ofexamples of compositions, those which contain proteins, glycoproteins orpeptides from the envelope protein corresponding to the entire 590-620region of the HIV-1_((VAU)) gp41 protein or to the parts of this regionwhich are specific for HIV-1_((VAU)) such as the peptides -TFIQN- or-WGCKNR-.

[0136] The invention also relates to compositions combining recombinantor synthetic HIV-1_((VAU)) proteins and/or glycoproteins and/or peptideswith proteins and/or glycoproteins and/or peptides from HIV-1 and/orHIV-2 and/or from another HIV-1 group O which are obtained by extractionor in lysates or by recombination or by chemical synthesis and/orpeptides which are derived from these proteins or glycoproteins andwhich are capable of being recognized by antibodies induced by the HIV-1and/or HIV-2 and/or HIV-1 group O virus.

[0137] The diagnostic compositions containing antigenic determinantscapable of being recognized by antibodies directed againstHIV-1_((VAU)), in particular the peptide compositions, may be includedin or combined with compositions or kits already available for detectinginfection by HIV-1 and/or HIV-2 retroviruses, so as to extend thedetection range of the kits to the detection of HIV-1 group Oretroviruses.

[0138] By way of nonlimiting examples:

[0139] either core proteins, particularly the gag, pol, HIV-1 and HIV-2proteins or peptides thereof, and HIV-1_((VAU)) envelope proteins orpeptides,

[0140] or HIV-1 envelope glycoproteins, HIV-2 envelope glycoproteins andHIV-1_((VAU)) envelope glycoproteins,

[0141] or mixtures of HIV-1 proteins and/or glycoproteins, HIV-2proteins and/or glycoproteins and HIV-1_((VAU)) envelope proteins and/orglycoproteins.

[0142] It is important to note that, although the antibodies frompatients infected with HIV-1 group O viruses react strongly with gag andpol antigens from HIV-1 group M viruses, their reactivity is practicallyzero with group M virus envelope antigens. It is therefore importantthat the composition of the present invention comprise at least oneprotein or one peptide of the HIV-1_((VAU)) envelope so that this viruscan be detected with certainty.

[0143] Such compositions, when used in diagnosis, consequently help thediagnosis of AIDS or of the symptoms associated with it, which extendover a broader spectrum of causative etiological agents. It goes withoutsaying that the use of diagnostic compositions which contain onlyHIV-1_((VAU)) envelope proteins and/or glycoproteins is nonethelessuseful for the more selective detection of the category of retroviruswhich may be held responsible for the disease.

[0144] Methods and Kits for the Diagnosis of Infections CausedEspecially by the HIV-1_((VAU)) Virus

[0145] The present invention relates to a method for the in vitrodiagnosis of infection caused by HIV viruses, etiological agents of AIDSand related syndromes, which comprises bringing a serum or anotherbiological medium, obtained from a patient or subject being subjected tothe diagnosis, into contact with a composition containing at least oneprotein, glycoprotein or peptide from HIV-1_((VAU)), and detecting apossible immunological reaction. Examples of such compositions weredescribed above.

[0146] Preferred methods involve, for example, immuno-fluorescence orELISA type immunoenzymic reactions. The detections may be effected bydirect or indirect immunofluorescence measurements or direct or indirectimmunoenzymic assays.

[0147] Such detections comprise for example:

[0148] depositing defined quantities of the extract or of the desiredantigenic compositions in accordance with the present invention into thewells of a microplate;

[0149] introducing into each well a serum, diluted or undiluted, whichis capable of containing the antibodies, the presence of which has to bedetected in vitro;

[0150] incubating the microplate;

[0151] carefully washing the microplate with an appropriate buffer;

[0152] introducing into the wells of the microplate specific labeledantibodies to human immunoglobulin, the labeling being performed with anenzyme chosen from those which are capable of hydrolyzing a substratesuch that the latter then undergoes modification of its absorption ofradiation, at least in a defined wavelength band, and

[0153] detecting, preferably in a comparative manner relative to acontrol, the extent of the hydrolysis of the substrate as a measure ofthe potential risks or of the actual presence of the infection.

[0154] The present invention also relates to kits or boxes for thediagnosis of HIV-1_((VAU)) virus infection, which comprise inparticular:

[0155] an extract, a more purified fraction, or a synthetic antigenderived from the types of viruses indicated above, this extract fractionor antigen being labeled, for example, radioactively, enzymically,fluorescently or otherwise,

[0156] antibodies to human immunoglobulins or a protein A (which isadvantageously attached to a support which is insoluble in water) suchas agarose beads for example) or microplate wells, and the like)

[0157] optionally, a sample of biological fluid or cells obtained from anegative control subject;

[0158] buffers and, where appropriate, substrates for visualizing thelabel.

[0159] The subject of the invention is also immunogenic compositionswhich are capable of inducing the formation of antibodies recognizingantigens which can be obtained by chemical synthesis or byrecombination.

[0160] Serology

[0161] The capacity of the serum antibodies from the patient infectedwith HIV-1_((VAU)) to react with HIV-1 antigenic preparations wasevaluated using various commercially available kits: Sanofi DiagnosticsPasteur, (Genelavia Mixt) Abbott, Wellcome, and Behring. The reactivityof these antibodies with various HIV-1 proteins was examined using theSanofi Diagnostics Pasteur Western-blot kit, following the proceduresrecommended by the manufacturer.

[0162] More precisely, the patient's serum was examined several timesusing HIV-1 specific ELISA kits. It was first tested and proved to bepositive in 1990, being noted 7.33 (this figure corresponds to the ratioof the measured OD to the background OD) with the Sanofi DiagnosticsPasteur kit, 3.50 with the Abbott kit and 2.70 with the Wellcome kit.During the use of reagents specific both for HIV-1 and HIV-2, the serumwas noted 1.42 with the Behring kit and 4.40 with the Wellcome kit.

[0163] The capacity of the patient's serum to react on different dateswith different HIV-1 structural proteins was studied using the HIV-1 LAVBLOT immunoblot assay, a test marketed by Sanofi Diagnostics Pasteur. Asshown in FIG. 5 with all the serum samples tested, only a very weakreactivity of the serum with the HIV-1 env proteins gp160 and gp120 wasnoted. However, the serum reacted strongly with the HIV-1 gag proteinsp55 (gag precursor) and p24 (CA), and with the pol products p66 (RT) andp34 (IN). By HIV-2 immunoblotting, only a very weak reactivity wasdetected with the gag p26.

[0164] This illustrates that the detection of antibodies specific forgroup O with commercially available serum diagnostic kits should becarefully controlled. Although serum antibodies from patients infectedwith group O viruses show strong cross-reactions with the group M gagand pol antigens, they show few or no reactions with the group Menvelope antigens. Consequently, it is possible to assume that asignificant proportion of these patients might not be detected usingsome kits based on group M envelope antigenic reagents. Indeed, in arecent preliminary study of several sera from patients infected withgroup O, it was found that the capacity to detect antibodies specificfor group O was very different depending on the detection kit used(Loussert-Ajaka, I., Ly, T. D., Chaix, M. L., Ingrand, D., Saragosti,S., Courroucé, A. M., Brun-Vézinet, F. and Simon, F. (1994). HIV-1/HIV-2seronegativity in HIV-1 subtype O infected patients. Lancet. 343,1393-1394.). This implies that a careful and extensive study of thereactivity of a large number of group O sera with all the diagnostickits available on the market is necessary. Compositions comprisingpolyclonal or monoclonal antibodies prepared from recombinant orsynthetic antigens from the HIV-1_((VAU)) virus.

[0165] The invention relates to a serum capable of being produced inanimals by inoculating them with HIV-1_((VAU)), particularly theantigenic epitopes of HIV-1_((VAU)) and more particularly the antigenicepitopes of the HIV-1_((VAT)) virus envelope protein. The inventionrelates more particularly to the polyclonal antibodies more specificallyoriented against each of the antigens, especially proteins orglycoproteins of the virus. It also relates to monoclonal antibodiesproduced by various techniques, these monoclonal antibodies beingrespectively oriented more specifically against the variousHIV-1_((VAU)) proteins, particularly the HIV-1_((VAU)) envelopeproteins.

[0166] These polyclonal or monoclonal antibodies can be used in variousapplications. There may be mentioned essentially their use forneutralizing the corresponding proteins, or even for inhibiting theinfectivity of the whole virus. They may also be used for example todetect viral antigens in biological preparations or to carry outprocedures for purifying the corresponding proteins and/orglycoproteins, for example during their use in affinity chromatographycolumns.

[0167] By way of example, anti-envelope antibodies or anti-gagantibodies are reagents which can be used in diagnosis, in particularfor the detection of HIV-1 group O particles by antigen capture ELISA.

[0168] The invention relates to antibodies directed against one or moreHIV-1_((VAU)) viral antigens produced from amino acid sequences ofHIV-1_((VAU)). Techniques for obtaining antibodies from antigenicepitopes similar to the antigenic epitopes of the HIV-1_((VAU)) virus ofthe present invention have been described previously.

[0169] The technique for the preparation of antibodies which isdescribed in the publication by Ulmer et al., 1993, may be used bypersons skilled in the art to prepare the antibodies of the presentinvention, the modifications which make it possible to adapt thistechnique to the antigens of the present invention forming part of theknowledge of persons skilled in the art.

[0170] Study of the Immunoreactivity of the vau Peptide

[0171] The immunoreactivity of the vau peptide was confirmed, afterexperimental ELISA plates had been prepared, according to a procedureestablished for a screening test for anti-HIV antibodies. This test isbased on the detection of a solid phase prepared with the peptide whichmimics the immunodominant epitope of the envelope glycoprotein of theHIV-1 group (or subgroup) O virus, VAU isolate. The implementation ofthe test was modeled on the procedure proposed by the Genelavia® Mixtkit, using the reagents in that kit.

[0172] The experimental data collated in the two tables of FIGS. 21 and22 show that:

[0173] a) the four sera taken from patients contaminated with the HIV-1group (or subgroup) O virus are very reactive with the vau peptide;

[0174] b) the ten sera supposedly taken from patients contaminated withthe HIV-1 (group or subgroup) O virus, among the 19 sera sent out by thePasteur Institute of Yacoundé, are also highly reactive with this samepeptide;

[0175] c) the sera (4 samples) taken from individuals contaminated withthe HIV-1 subtype B virus (in the acute phase) are not reactive with thevau peptide;

[0176] d) the sera taken from asymptomatic blood donors (48 samplestested) are not reactive with the vau peptide; These experimental data,although limited (in view of the paupicity of HIV-1 group (or subgroupO) antibody-positive samples), bear witness to the sensitivity andspecificity of the peptide selected.

[0177] From the above text, it follows that the invention also relatesto the detection of the HIV-1_((VAU)) virus or of a variant by virtue ofthe use of the antibodies described above in a method involving variousstages, these stages being specifically intended to reveal thecharacteristic properties of the HIV-1_((VAU)) virus.

[0178] The invention also relates to the detection of the HIV-1_((vau))virus by molecular hybridization.

[0179] Generally, this method for detecting the HIV-1_((VAU)) virus or avariant in serum samples or samples of other biological fluids ortissues, obtained from patients liable to be carriers of theHIV-1_((VAU)) virus, comprises the following stages:

[0180] the manufacture of at least one optionally labeled probe;

[0181] at least one hybridization stage performed under conditionspermitting hybridization by bringing the nucleic acid of the suspectpatient's sample into contact with said labeled probe and optionallyimmobilizing the complex formed on an appropriate solid support,

[0182] where appropriate, washing said solid support with a suitablewashing solution,

[0183] the detection of said complex and therefore of the presence orotherwise of the HIV-1_((VAU)) virus by an appropriate detection methodknown to those skilled in the art.

[0184] In another preferred embodiment of the method according to theinvention, the abovementioned hybridization is performed undernonstringent conditions and the membrane is washed under conditionsadapted to those for the hybridization.

[0185] By using serology or a gene amplification technique such asspecific Polymerase Chain Reaction (PCR), the extent of the HIV-1 groupO epidemic was precisely evaluated. It was found that 5 to 10% ofpatients infected with HIV-1 in Cameroon are in fact infected with groupO viruses. However, apart from the virus isolate described here, thepropagation of the group O virus outside West Central Africa has notbeen documented. The patient from whom HIV-1_((VAU)) was isolated hasalways lived in France and has never traveled to Africa. Up until now,we have no precise proof relating to the origin of her infection, butthis case indicates that a degree of propagation of the group O virusesin Europe has already occurred.

[0186] The invention also relates to a process of detection anddiscrimination, in a biological sample, between antibodiescharacteristic of an HIV-1 group (or subgroup) O retrovirus andantibodies characteristic of a retrovirus of the HIV-1-M type,characterized by placing this biological sample in contact with apeptide which does not react with the antibodies characteristic of aretrovirus of the HIV-1-M type, in particular one chosen from thepeptides (1), (2), (3), (4), (5a), (5b), (9) and (10) described above.

[0187] Also, the invention relates to a process of detection anddiscrimination, in a biological sample, between antibodiescharacteristic of an HIV-1 group (or subgroup) O retrovirus andantibodies characteristic of a retrovirus of the HIV-1-M type,characterized by placing this biological sample in contact with thepeptide derived from one of the HIV-1 M viruses taken into considerationin FIGS. 8 and 9 and homologous to a peptide chosen from the peptides(1), (2), (3), (4), (5a), (5b), (9) and (10), the sequence of thishomologous peptide resulting from vertical alignments of its ownsuccessive amino acids which are themselves contained in the pertinentpeptide sequence relative to the corresponding HIV-1-M virus andrepresented in FIG. 8 or 9 with the successive amino acids of thesequence of the peptide chosen, as also follows from FIG. 8 or 9.

[0188] According to the present invention, the process of detection anddiscrimination between infection by an HIV-1 group (or subgroup) Oretrovirus and an HIV-1 subgroup M retrovirus is characterized byplacing serum, obtained from individuals subjected to an AIDS diagnostictest, in contact, in particular, with the peptide RILAVERY.

[0189] In addition, the process for detection of infection due either toan HIV-1 subgroup O or HIV-1 subgroup M retrovirus is characterized bythe use of mixtures of two categories of peptides, those of the firstcategory corresponding to the peptides (1), (2), (3), (4), (5a), (5b),(9) and (10).

[0190] Moreover, the process for discrimination between an infection dueto an HIV-1-O DUR retrovirus and an infection due to another type ofHIV-1-O retrovirus, is characterized by placing the biological testsample in contact with any of the peptides (11) to (15) or of thepeptides (17) to (20).

[0191] Alternatively, it is a process for discrimination between aninfection by an HIV-1 group (or subgroup) O retrovirus and an HIV-1subgroup M retrovirus, using a serine protease whose cleaving action iscarried out on an SR dipeptide, and comprising the detection of acleavage or of a non-cleavage of the V3 loop of gp120 of the retrovirus,depending on whether this retrovirus is an HIV-1 group (or subgroup) Oretrovirus or an HIV-1 subgroup M retrovirus.

[0192] The invention also relates to a composition for detection anddiscrimination, in a biological sample, between an HIV-1 subgroup Mretrovirus and an HIV-1 group (or subgroup) O retrovirus, comprising amixture of two categories of peptides, the first being in particularthose identified (1), (2), (3), (4), (5a), (5b), (9) and (10)

[0193] Monoclonal antibodies specific for the sequences of each of thepeptides (1) to (20) also fall within the scope of the presentinvention.

[0194] The invention also relates to a plasmid chosen from those whichwere deposited at the CNCM on 24 February 1995 under the referencesI-1548, I-1549 and I-1550.

[0195] The invention is also directed towards nucleic acids containing asequence which codes for each of the peptides (1) to (20) defined in thepresent invention.

[0196] Among the preferred nucleic acid sequences, the nucleotidesequences represented in FIGS. 10, 11 or 12 will be chosen.

[0197] The invention also relates to vectors containing a nucleic acidas defined above.

[0198] The invention is also directed towards cells liable to containany one of said nucleic acids or of said vectors.

[0199] The present invention also relates to a virus such as thatdeposited on Feb. 23, 1995 at the CNCM under the reference I-1542.

[0200] A virus also falling within the scope of the invention is a virusof the same group as the above, characterized in that consensus peptidesof this virus are recognized by antibodies which specifically recognizea polypeptide or a peptide defined above.

[0201] The genomic RNA of this virus also falls within the scope of theinvention.

[0202] Also falling within the scope of the invention is a box or kitfor detection of antibodies in the serum or any other biological samplefrom a patient liable to be infected with a human retrovirus of the HIVtype, characterized in that it comprises:

[0203] at least one polypeptide or one peptide having, in particular, asits sequence one of the sequences (1) to (20) described above.

[0204] means allowing the reaction for formation of the immunologicalcomplex between the polypeptide(s) or the peptide(s) and the antibodieswhich may be present in the biological sample to be tested, for exampleone or more incubation buffers, if needed,

[0205] a negative control sample,

[0206] means for visualizing the antigen/antibody complex formed.

[0207] Also according to the invention, this kit contains, in addition,at least one consensus peptide or polypeptide derived from another HIVstrain or from a peptide or polypeptide comprising

[0208] either an amino acid sequence which is separate from the sequenceof this polypeptide or peptide, in which one or more amino acids arereplaced with other amino acids, with the proviso that the peptide orpolypeptide retains its reactivity with an antiserum against theconsensus peptide or polypeptide,

[0209] or an amino acid sequence in which one or more amino acids havebeen deleted or added, with the proviso that the peptide or polypeptideretains its reactivity with an antiserum against the consensus peptideor polypeptide.

[0210] Preferably, a kit according to the invention will contain, inaddition, at least one peptide or polypeptide derived from another HIVstrain, preferably the HIV-LAI strain.

[0211] The invention also relates to a polypeptide composition for thein vitro diagnosis of an infection due to a retrovirus according to theinvention, or to one of its variants, this diagnosis being made on abiological sample liable to contain antibodies formed after saidinfection. This composition is characterized in that it comprises atleast one of the peptides (1) to (20).

[0212] The biological sample may consist in particular of blood, plasma,serum or any other biological extract. The above compositions may beused for the detection of antibodies in one of the abovementionedbiological samples.

[0213] The invention is therefore also directed toward a method for thein vitro diagnosis of an infection due specifically to a retrovirus ofthe HIV type, characterized by the steps of:

[0214] placing a biological sample, which is liable to containantibodies produced after an infection by an HIV-1 group (or subgroup) Oretrovirus, in contact with a peptide as defined above, or with apeptide composition as defined above, under suitable conditions whichallow the formation of an immunological complex of the antigen/antibodytype,

[0215] detection of the possible presence of the complex.

[0216] The invention moreover relates to an immunogenic composition,characterized in that it comprises at least one peptide in combinationwith a pharmaceutical vehicle which is acceptable for making upvaccines.

[0217] The invention also relates to a process for the preparation ofcapsid proteins and gp41 and gp120 glycoproteins of a retroviral strainaccording to the invention, the process being characterized by thefollowing steps:

[0218] lysis of the cells infected with an HIV-1 retrovirus according tothe invention and separation of the supernatant and of the infectedcells or lysis of the viral pellets prepared by centrifugation,

[0219] deposition of the cell extract and/or of the viral extract on animmunoadsorbant containing purified antibodies, which are obtained fromthe serum of an individual infected by a retrovirus according to theinvention and advantageously attached to a suitable support, said serumof the infected individual having the capacity to react strongly withenvelope proteins of the virus according to the invention,

[0220] incubation in the presence of a buffer and for a sufficientlylong period to obtain the formation of an antigen/antibody immunologicalcomplex,

[0221] washing of the immunoadsorbant with a buffer in order to removethe molecules not retained on the support,

[0222] recovery of the desired antigenic proteins.

[0223] According to a first embodiment of this preparation process, theseparation and the recovery of the capsid proteins and of the gp41 andgp120 glycoproteins of HIV-1 DUR are carried out by electrophoresis andby electroreduction of the proteins.

[0224] According to another embodiment of this preparation process, theproteins are recovered by:

[0225] elution of the proteins attached to the above immunoadsorbant,

[0226] purification of the products thus eluted on a chromatographycolumn containing, attached to the separation support, antibodies whichrecognize the capsid proteins and the gp41 and gp120 glycoproteins ofHIV-1 group (or subgroup) O DUR.

[0227] Also falling within the scope of the invention is a process forthe production of a peptide or polypeptide according to the invention,this peptide or polypeptide being obtained

[0228] either by expression of a nucleic acid of the invention,

[0229] or by chemical synthesis, by addition of amino acids until thispeptide or this polypeptide is obtained.

[0230] The standard principles and processes of genetic engineering maybe used here (“Molecular cloning”, Sambrook, Fritsch, Maniatis, CSH1989).

[0231] Also falling within the scope of the invention is a process forthe production of a nucleic acid defined above, which may be producedeither by isolation from a virus of the invention, or by chemicalsynthesis, or by using techniques of in vitro amplification of nucleicacids from specific primers.

[0232] Oligonucleotide primers also according to the invention have asequence consisting of at least eight consecutive nucleotides of thefollowing nucleotide sequences: ATT CCA ATA CAC TAT TGT GCT CCA-3′ AAAGAA TTC TCC ATG ACT GTT AAA-3′ GGT ATA GTG CAA CAG CAG GAC AAC-3′ AGAGGC CCA TTC ATC TAA CTC-3′

[0233] These primers may be used in a gene amplification process, forexample by PCR or an equivalent technique, of a nucleotide sequencecoding for a peptide of the invention. Tests carried out with theseprimers gave conclusive results.

[0234] Also, the invention relates to a kit allowing amplification byPCR or an equivalent technique described above.

[0235] Also falling within the scope of the present invention is aprocess for detection of the presence, in a biological sample, ofnucleic acid(s) characteristic of an HIV-1 group (or subgroup) O DURretrovirus, including a retrovirus according to the invention. Thisprocess comprises a placing in contact of a cDNA formed from RNA(s)contained in this biological sample, under conditions allowing thehybridization of this CDNA with the retroviral genome, and the executionof a gene amplification on this viral sample.

[0236] The invention also relates to a viral lysate as obtained by lysisof cells infected with a virus according to the invention.

[0237] A protein extract from an HIV-1_((DUR)) (or HIV-1_((VAU))) straincontaining in particular a peptide or a polypeptide as defined abovealso falls within the scope of the invention.

[0238] The invention relates to specific peptides obtained from thestructure of HIV-1 group (or subgroup) O DUR or from variants of thisretrovirus and which make it possible

[0239] either to discriminate, depending on the case,

[0240] globally between HIVs-1 belonging to the category O and HIVs-1belonging to the category M,

[0241] or, more specifically, between viruses belonging to the subgroupcharacteristic of the HIV-1 group (or subgroup) O DUR and other virusesof the subgroup O,

[0242] or, on the other hand, to recognize most, if not all, of theretroviruses both of the group (or subgroup) O and of the subgroup M.

[0243] Also falling within the scope of the invention are thecorresponding peptides obtained from corresponding structural proteinsof other HIV-1 group (or subgroup) O or HIV-1 subgroup M viruses, inparticular those derived from the GAG, gp120 and gp41 structuralproteins whose parts are indicated in the diagrams, these homologouspeptides ensuing from their being placed in alignment, as also resultsfrom the diagrams with the peptides obtained from the HIV-1 group (orsubgroup) O DUR, more particularly identified in the present text.

[0244] Similarly, certain homologous peptides may be used in those testswhich make it possible to carry out the abovementioned discriminations,it being understood in this case that they are then used in place of thecorresponding peptides, derived from the GAG, gp120 and gp41 structuralproteins.

[0245] Determination of Oligonucleotides Specific for the O Group

[0246] Using the VAU sequence and its correlation with the MVP5180 andANT70 sequences, oligonucleotide primers were defined which endeavor tobe specific for the subgroup O in its entirety for the V3 region and thegp41 region. These primers made it possible to amplify the DUR strainand consequently constituted one solution to the amplification problemencountered. The position and the sequence of these HIV subgroup Oprimers are represented in FIG. 13. These primers make it possible toobtain an amplification band which is visible on staining with ethidiumbromide, with a single step of 30 cycles of PCR. Partial sequences wereobtained:

[0247] GAG: 513 base pairs (171 amino acids)=Seq ID No. 9

[0248] gp120 V3 loop: 525 base pairs (75 amino acids)=Seq ID No. 10

[0249] gp41 immunodominant region: 312 base pairs (104 amino acids)=SeqID No. 11.

[0250] Nucleotide (FIG. 15) and protein (FIG. 16) comparisons of the DURsequences with the MVP5180, ANT and VAU sequences for the O subgroup,LAI for the HIV-1 consensus sequence, representative African HIV-1 MALsequence, and CPZ for the CIV of the Gabonese chimpanzee, show that DURis as remote from the other published HIV-1 group (or subgroup) Ostrains as the latter are from each other.

[0251] The differences are less in the GAG region and maximal in theregion of the V3 loop of gp120, where the protein comparison reaches adifference of 40% (FIG. 16). The phylogenic trees confirm, on the onehand, that the DUR strain forms part of the O subgroup, and, on theother hand, the importance of the differences between the various Ostrains described, without, however, subtype branching emerging clearly(FIG. 17).

[0252] Comparison of the GAG Sequences:

[0253] On comparison of the GAG sequence obtained with the other two Ostrains published, ANT70 and MVP5180, as well as with the representativesequences of the M group (FIG. 8), it was possible to observe that an Oconsensus sequence exists in several regions, which is distinct from theM consensus sequence in the same regions. Two hypervariable regions,which are more variable for O than for M, and a few point differencesfor one or the other strain may also be found. Nevertheless, the regionsSPRT . . . SEGA, MNAI . . . KEVIN, GPLPP . . . QQEQI and VGD . . . SPVappear to discriminate between the O consensus sequence and the Mconsensus sequence.

[0254] The regions QQA and LWTTRAGNP are hypervariable regions. TheHIV-1 group (or subgroup) O DUR strain is conspicuously different inthree positions with respect to the M and O consensus sequence (L for Iand twice for E) and takes a specific amino acid in three isolatedhypervariable positions, V position L9; A position A77; L position 110.

[0255] In addition, it is possible to define in the GAG region segmentscommon to the O group and to the M group, such as SPRTLNAWVK, GSDIAGTTSTand QGPKEPFRDYVDRF.

[0256] Comparison of the Sequences of the V3 Loop

[0257] This comparative study revealed considerable differences of up to56% for protein differences with the HIV-1 subgroup M consensussequence, and 35 to 42% with the other HIV-1 group (or subgroup) Oconsensus sequences.

[0258] The alignments of peptide sequences in the regions of the V3 loopof gp120 and in the immunodominant region of gp41 is given in FIG. 9.The sequence of the interior of the V3 loop of the DUR strain differssubstantially from that of the HIV-1 subgroup M consensus sequence. Itshares the motif GPMAWYSM with the VAU and ANT70 strains but not withthe MVP strain, which has two substitutions: R for A and R for Y.

[0259] The left and right parts of the rest of the V3 loop are markedlydifferent from all the other HIVs known and do not leave it possible toimagine other cross-reactivities. Furthermore, the V3 loop of DUR is oneamino acid longer than the other O sequences, which are themselvesanother one amino acid longer than the sequences of the HIV-1 M group.

[0260] Comparison of the Alignments Concerning the Immunodominant Regionof gp41

[0261] The “mini loop” of the DUR strain, having the sequence CRGKAIC,proved to be very specific for this strain: it might constitute anepitope (see FIG. 9). In addition, this sequence might be likely to beinvolved in the modification of the conditions of unfolding of the gp41glycoprotein, and consequently in the infectiousness of the strain.

[0262] A long sequence of 11 amino acids flanking this loop on the leftis identical to the VAU sequence. A polymorphism of the DUR strain maybe noted for an S or T position according to the clones analyzed.

[0263] Corresponding peptides obtained from other known retroviralstrains are also represented in FIG. 9.

[0264] The DUR strain also makes it possible to define an HIV subgroup Oconsensus sequence of the gp41 region, several sufficiently longhomologous regions of which might be used. These homologous regions are,inter alia: RL*ALET, QNQQ, LWGC and CYTV (*representing a variable aminoacid).

[0265] Serological Correlations:

[0266] The anti-DUR antiserum does not react with the peptides of the V3loop of the HIV-1-M consensus sequence, of HIV-1 MAL, of HIV-1 CPZ or ofHIV-1 group (or subgroup) O MVP5180 but does, however, react with thepeptide of the V3 loop of HIV-1-O ANT70. As regards the gp41immunodominant region, this does not react with the “standard” HIV-1subgroup M consensus sequence, but does, however, react, weakly butsurprisingly, with the HIV-1 subgroup M right-extended consensussequence.

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1 103 23 base pairs nucleic acid single linear Other nucleic acid /desc= “oligonucleotide” 1 AGTGGATWYA TAGAAGCAGA AGT 23 20 base pairs nucleicacid single linear Other nucleic acid /desc = “oligonucleotide” 2ACTGCYCCTT CHCCTTTCCA 20 28 base pairs nucleic acid single linear Othernucleic acid /desc = “oligonucleotide” 3 GCTCTAGATG GGGATCTCCC ATGGCAGG28 30 base pairs nucleic acid single linear Other nucleic acid /desc =“oligonucleotide” 4 GCTCTAGATC AGGGAAGAAT CCCTGAGTGT 30 7 amino acidsamino acid single linear peptide 5 Cys Lys Asn Arg Leu Ile Cys 1 5 26amino acids amino acid single linear peptide 6 Arg Leu Leu Ala Leu GluThr Phe Ile Gln Asn Trp Trp Leu Leu Asn 1 5 10 15 Leu Trp Gly Cys LysAsn Arg Leu Ile Cys 20 25 37 amino acids amino acid single linearpeptide 7 Arg Ala Arg Leu Leu Ala Leu Glu Thr Phe Ile Gln Asn Gln GlnLeu 1 5 10 15 Leu Asn Leu Trp Gly Cys Lys Asn Arg Leu Ile Cys Tyr ThrSer Val 20 25 30 Lys Trp Asn Lys Thr 35 6 amino acids amino acid singlelinear peptide 8 Ala His Pro Gln Gln Ala 1 5 9 amino acids amino acidsingle linear peptide 9 Leu Trp Thr Thr Arg Ala Gly Asn Pro 1 5 32 aminoacids amino acid single linear peptide 10 Ser Pro Arg Thr Leu Asn AlaTrp Val Lys Ala Val Glu Glu Lys Ala 1 5 10 15 Phe Asn Pro Glu Ile IlePro Met Phe Met Ala Leu Ser Glu Gly Ala 20 25 30 20 amino acids aminoacid single linear peptide 11 Met Leu Asn Ala Ile Gly Gly His Gln GlyAla Leu Gln Val Leu Lys 1 5 10 15 Glu Val Ile Asn 20 27 amino acidsamino acid single linear peptide 12 Gly Pro Leu Pro Pro Gly Gln Ile ArgGlu Pro Thr Gly Ser Asp Ile 1 5 10 15 Ala Gly Thr Thr Ser Thr Gln GlnGlu Gln Ile 20 25 30 amino acids amino acid single linear peptide 13 IlePro Val Gly Asp Ile Tyr Arg Lys Trp Ile Val Leu Gly Leu Asn 1 5 10 15Lys Met Val Lys Met Tyr Ser Pro Val Ser Ile Leu Asp Ile 20 25 30 21amino acids amino acid single linear peptide 14 Gln Gly Pro Lys Glu ProPhe Arg Asp Tyr Val Asp Arg Phe Tyr Lys 1 5 10 15 Thr Lys Leu Ala Glu 2010 amino acids amino acid single linear peptide 15 Ser Pro Arg Thr LeuAsn Ala Trp Val Lys 1 5 10 10 amino acids amino acid single linearpeptide 16 Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr 1 5 10 14 amino acidsamino acid single linear peptide 17 Gln Gly Pro Lys Glu Pro Phe Arg AspTyr Val Asp Arg Phe 1 5 10 4 amino acids amino acid single linearpeptide 18 Asn Pro Glu Ile 1 16 amino acids amino acid single linearpeptide 19 Ala Val Glu Glu Lys Ala Phe Asn Pro Glu Ile Ile Pro Met PheMet 1 5 10 15 9 amino acids amino acid single linear peptide 20 Ile GlyGly His Gln Gly Ala Leu Gln 1 5 8 amino acids amino acid single linearpeptide 21 Arg Glu Pro Thr Gly Ser Asp Ile 1 5 9 amino acids amino acidsingle linear peptide 22 Ile Asn Asp Glu Ala Ala Asp Trp Asp 1 5 5 aminoacids amino acid single linear peptide 23 Lys Glu Ile Lys Ile 1 5 8amino acids amino acid single linear peptide 24 Glu Arg Glu Gly Lys GlyAla Asn 1 5 14 amino acids amino acid single linear peptide 25 Cys ValArg Pro Gly Asn Asn Ser Val Lys Glu Ile Lys Ile 1 5 10 12 amino acidsamino acid single linear peptide 26 Gln Ile Glu Arg Glu Gly Lys Gly AlaAsn Ser Arg 1 5 10 38 amino acids amino acid single linear peptide 27Cys Val Arg Pro Gly Asn Asn Ser Val Lys Glu Ile Lys Ile Gly Pro 1 5 1015 Met Ala Trp Tyr Ser Met Gln Ile Glu Arg Glu Gly Lys Gly Ala Asn 20 2530 Ser Arg Thr Ala Phe Cys 35 8 amino acids amino acid single linearpeptide 28 Gly Pro Met Ala Trp Tyr Ser Met 1 5 14 amino acids amino acidsingle linear peptide 29 Arg Leu Leu Ala Leu Glu Thr Leu Met Gln Asn GlnGln Leu 1 5 10 23 amino acids amino acid single linear peptide 30 LeuAsn Leu Trp Gly Cys Arg Gly Lys Ala Ile Cys Tyr Thr Ser Val 1 5 10 15Gln Trp Asn Glu Thr Trp Gly 20 6 amino acids amino acid single linearpeptide 31 Cys Arg Gly Lys Ala Ile 1 5 5 amino acids amino acid singlelinear peptide 32 Ser Val Gln Trp Asn 1 5 29 amino acids amino acidsingle linear peptide 33 Arg Leu Leu Ala Leu Glu Thr Leu Met Gln Asn GlnGln Leu Leu Asn 1 5 10 15 Trp Gly Cys Arg Gly Lys Ala Ile Cys Tyr ThrSer 20 25 24 amino acids amino acid single linear peptide 34 Gln Asn GlnGln Leu Leu Asn Leu Trp Gly Cys Arg Gly Lys Ala Ile 1 5 10 15 Cys TyrThr Ser Val Gln Trp Asn 20 8 amino acids amino acid single linearpeptide 35 Arg Ile Leu Ala Val Glu Arg Tyr 1 5 6 amino acids amino acidsingle linear peptide 36 Ser Gly Lys Leu Ile Cys 1 5 6 amino acids aminoacid single linear peptide 37 Gly Pro Gly Arg Ala Phe 1 5 6 amino acidsamino acid single linear peptide 38 Gly Pro Met Ala Trp Tyr 1 5 6 aminoacids amino acid single linear peptide 39 Gly Pro Met Arg Trp Arg 1 5 5amino acids amino acid single linear peptide 40 Thr Phe Ile Gln Asn 1 56 amino acids amino acid single linear peptide 41 Trp Gly Cys Lys AsnArg 1 5 24 base pairs nucleic acid single linear Other nucleic acid/desc = “oligonucleotide” 42 ATTCCAATAC ACTATTGTGC TCCA 24 24 base pairsnucleic acid single linear Other nucleic acid /desc = “oligonucleotide”43 AAAGAATTCT CCATGACTGT TAAA 24 24 base pairs nucleic acid singlelinear Other nucleic acid /desc = “oligonucleotide” 44 GGTATAGTGCAACAGCAGGA CAAC 24 21 base pairs nucleic acid single linear Othernucleic acid /desc = “oligonucleotide” 45 AGAGGCCCAT TCATCTAACT C 21 526amino acids amino acid single linear protein 46 Met Thr Ala Ile Met LysAla Met Gly Lys Arg Asn Arg Lys Leu Gly 1 5 10 15 Ile Trp Cys Leu IleLeu Ala Leu Ile Ile Pro Cys Leu Ser Cys Asn 20 25 30 Gln Leu Tyr Ala ThrVal Tyr Ser Gly Val Pro Val Trp Glu Asp Ala 35 40 45 Lys Pro Thr Leu PheCys Ala Ser Asp Ala Asn Leu Thr Ser Thr Glu 50 55 60 Gln His Asn Ile TrpAla Thr Gln Ala Cys Val Pro Thr Asp Pro Ser 65 70 75 80 Pro Asn Glu TyrGlu Leu Lys Asn Val Thr Gly Lys Phe Asn Ile Trp 85 90 95 Lys Asn Tyr IleVal Asp Gln Met His Glu Asp Ile Ile Asp Leu Trp 100 105 110 Asp Gln SerLeu Lys Pro Cys Val Gln Met Thr Phe Leu Cys Val Gln 115 120 125 Met AsnCys Thr Asp Ile Lys Asn Ser Ile Asn Thr Thr Asn Ser Pro 130 135 140 LeuAsn Ser Asn Asn Thr Lys Glu Val Lys Gln Cys Asp Phe Asn Val 145 150 155160 Thr Thr Val Leu Lys Asp Lys Gln Glu Lys Lys Gln Ala Leu Phe Tyr 165170 175 Val Thr Asp Leu Val Lys Ile Asn Ala Thr Ser Asn Glu Thr Met Tyr180 185 190 Arg Leu Ile Asn Cys Asn Ser Thr Thr Ile Arg Gln Ala Cys ProLys 195 200 205 Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro AlaGly Cys 210 215 220 Ala Ile Phe Lys Cys Asn Glu Thr Gly Phe Asn Gly ThrGly Leu Cys 225 230 235 240 Lys Asn Val Thr Val Val Thr Cys Thr His GlyIle Lys Pro Thr Val 245 250 255 Ser Thr Gln Leu Ile Leu Asn Gly Thr LeuSer Lys Gly Asn Ile Thr 260 265 270 Ile Met Gly Lys Asn Ile Ser Asp SerGly Glu Asn Ile Leu Ile Thr 275 280 285 Leu Asn Thr Asn Ile Thr Ile AlaCys Glu Arg Pro Gly Asn Gln Thr 290 295 300 Ile Gln Lys Ile Met Ala GlyPro Met Ala Trp Tyr Ser Met Ala Leu 305 310 315 320 Ser Asn Thr Lys GlyAsp Thr Arg Ala Ala Tyr Cys Asn Tyr Ser Ala 325 330 335 Thr Asp Trp AsnLys Ala Leu Lys Asn Ile Thr Glu Arg Tyr Leu Glu 340 345 350 Leu Val GluTyr Asn Gln Thr Asp Val Thr Met Lys Phe Gly Asn His 355 360 365 Ser GlyGlu Asp Ala Glu Val Thr Asn Phe Phe Phe Asn Cys His Gly 370 375 380 GluPhe Phe Tyr Cys Asn Thr Asn Arg Leu Phe Asn His Thr Phe Ser 385 390 395400 Cys Lys Lys Asn Met Thr Asn Asn Lys Ile Asn Cys Thr Asn Ile Ser 405410 415 Asn Asn Ser Asn Gly Thr Gln Ala Ile Pro Cys Arg Leu Arg Gln Val420 425 430 Val Arg Asp Trp Met Arg Gly Gly Ser Gly Leu Tyr Ala Pro ProIle 435 440 445 Pro Gly Asn Leu Val Cys Arg Ser Asn Ile Thr Gly Met IleLeu Gln 450 455 460 Leu Asp Thr Pro Trp Asn Lys Thr His Pro Asn Ser ThrThr Leu Arg 465 470 475 480 Pro Gly Gly Gly Asp Met Lys Asp Ile Trp ArgThr Gln Leu Phe Lys 485 490 495 Tyr Lys Val Val Arg Val Lys Pro Phe SerVal Ala Pro Thr Lys Ile 500 505 510 Ala Arg Pro Thr Ile Gly Thr Arg SerHis Arg Glu Lys Arg 515 520 525 351 amino acids amino acid single linearprotein 47 Ala Ala Gly Leu Ala Met Leu Phe Leu Gly Ile Leu Ser Ala AlaGly 1 5 10 15 Ser Thr Met Gly Ala Ala Ala Thr Ala Leu Thr Val Arg ThrGln His 20 25 30 Leu Ile Lys Gly Ile Val Gln Gln Gln Asp Asn Leu Leu ArgAla Ile 35 40 45 Gln Ala Gln Gln His Leu Leu Arg Pro Ser Val Trp Gly IleArg Gln 50 55 60 Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr Phe Ile Gln AsnGln Gln 65 70 75 80 Leu Leu Asn Leu Trp Gly Cys Lys Asn Arg Leu Ile CysTyr Thr Ser 85 90 95 Val Lys Trp Asn Lys Thr Trp Gly Gly Asp Asn Glu SerIle Trp Asp 100 105 110 Glu Leu Thr Trp Gln Gln Trp Asp Gln Gln Ile AsnAsn Val Ser Ser 115 120 125 Phe Ile Tyr Glu Lys Ile Gln Glu Ala Gln GluGln Gln Glu Lys Asn 130 135 140 Glu Lys Glu Leu Leu Glu Leu Asp Glu TrpAla Ser Ile Trp Asn Trp 145 150 155 160 Leu Asp Ile Thr Lys Trp Leu TrpTyr Ile Lys Ile Ala Ile Ile Ile 165 170 175 Val Gly Ala Leu Ile Gly ValArg Val Val Met Ile Val Leu Asn Leu 180 185 190 Val Lys Asn Ile Arg GlnGly Tyr Gln Pro Leu Ser Leu Gln Ile Pro 195 200 205 Ile Gln Gln Gln AlaGlu Val Gly Thr Pro Gly Gly Thr Gly Glu Gly 210 215 220 Gly Gly Asp GluAsp Arg Arg Arg Trp Thr Pro Leu Pro Gln Gly Phe 225 230 235 240 Leu HisLeu Leu Tyr Thr Asp Leu Arg Thr Ile Ile Leu Trp Ile Tyr 245 250 255 HisLeu Leu Ser Asn Leu Ala Ser Glu Ile Gln Lys Leu Ile Arg His 260 265 270Leu Gly Leu Gly Leu Trp Ile Ile Gly Gln Arg Thr Ile Glu Ala Cys 275 280285 Arg Leu Phe Lys Ala Ile Ile Gln Tyr Trp Leu Gln Glu Leu Gln Thr 290295 300 Ser Ala Thr Asn Leu Leu Asp Thr Val Ala Val Ala Val Ala Asn Trp305 310 315 320 Thr Asp Ser Thr Ile Leu Gly Ile Gln Ser Ile Gly Arg GlyIle Leu 325 330 335 Asn Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu Arg LeuLeu Leu 340 345 350 516 amino acids amino acid single linear protein 48Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Lys 1 5 1015 Trp Gly Thr Met Leu Leu Gly Ile Leu Met Ile Cys Ser Ala Thr Glu 20 2530 Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala 35 4045 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 5560 Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 7075 80 Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp 8590 95 Lys Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp100 105 110 Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys ValSer 115 120 125 Leu Lys Cys Thr Asp Leu Gly Asn Ala Thr Asn Thr Asn SerSer Asn 130 135 140 Thr Asn Ser Ser Ser Gly Glu Met Met Met Glu Lys GlyGlu Ile Lys 145 150 155 160 Asn Cys Ser Phe Asn Ile Ser Thr Ser Ile ArgGly Lys Val Gln Lys 165 170 175 Glu Tyr Ala Phe Phe Tyr Lys Leu Asp IleIle Pro Ile Asp Asn Asp 180 185 190 Thr Thr Ser Tyr Thr Leu Thr Ser CysAsn Thr Ser Val Ile Thr Gln 195 200 205 Ala Cys Pro Lys Val Ser Phe GluPro Ile Pro Ile His Tyr Cys Ala 210 215 220 Pro Ala Gly Phe Ala Ile LeuLys Cys Asn Asn Lys Thr Phe Asn Gly 225 230 235 240 Thr Gly Pro Cys ThrAsn Val Ser Thr Val Gln Cys Thr His Gly Ile 245 250 255 Arg Pro Val ValSer Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu 260 265 270 Glu Glu ValVal Ile Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr 275 280 285 Ile IleVal Gln Leu Asn Gln Ser Val Glu Ile Asn Cys Thr Arg Pro 290 295 300 AsnAsn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg 305 310 315320 Ala Phe Val Thr Ile Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys 325330 335 Asn Ile Ser Arg Ala Lys Trp Asn Ala Thr Leu Lys Gln Ile Ala Ser340 345 350 Lys Leu Arg Glu Gln Phe Gly Asn Asn Lys Thr Ile Ile Phe LysGln 355 360 365 Ser Ser Gly Gly Asp Pro Glu Ile Val Thr His Ser Phe AsnCys Gly 370 375 380 Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe AsnSer Thr Trp 385 390 395 400 Phe Asn Ser Thr Trp Ser Thr Glu Gly Ser AsnAsn Thr Glu Gly Ser 405 410 415 Asp Thr Ile Thr Leu Pro Cys Arg Ile LysGln Phe Ile Asn Met Trp 420 425 430 Gln Glu Val Gly Lys Ala Met Tyr AlaPro Pro Ile Ser Gly Gln Ile 435 440 445 Arg Cys Ser Ser Asn Ile Thr GlyLeu Leu Leu Thr Arg Asp Gly Gly 450 455 460 Asn Asn Asn Asn Gly Ser GluIle Phe Arg Pro Gly Gly Gly Asp Met 465 470 475 480 Arg Asp Asn Trp ArgSer Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile 485 490 495 Glu Pro Leu GlyVal Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln 500 505 510 Arg Glu LysArg 515 345 amino acids amino acid single linear protein 49 Ala Val GlyIle Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly 1 5 10 15 Ser ThrMet Gly Ala Arg Ser Met Thr Leu Thr Val Gln Ala Arg Gln 20 25 30 Leu LeuSer Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile 35 40 45 Glu AlaGln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln 50 55 60 Leu GlnAla Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln 65 70 75 80 LeuLeu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala 85 90 95 ValPro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp 100 105 110Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr 115 120125 Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys 130135 140 Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn145 150 155 160 Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys Ile PheIle Met 165 170 175 Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe AlaVal Leu Ser 180 185 190 Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro LeuSer Phe Gln Thr 195 200 205 His Leu Pro Thr Pro Arg Gly Pro Asp Arg ProGlu Gly Ile Glu Glu 210 215 220 Glu Gly Gly Glu Arg Asp Arg Asp Arg SerIle Arg Leu Val Asn Gly 225 230 235 240 Ser Leu Ala Leu Ile Trp Asp AspLeu Arg Ser Leu Cys Leu Phe Ser 245 250 255 Tyr His Arg Leu Arg Asp LeuLeu Leu Ile Val Thr Arg Ile Val Glu 260 265 270 Leu Leu Gly Arg Arg GlyTrp Glu Ala Leu Lys Tyr Trp Trp Asn Leu 275 280 285 Leu Gln Tyr Trp SerGln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu 290 295 300 Asn Ala Thr AlaIle Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu 305 310 315 320 Val ValGln Gly Ala Cys Arg Ala Ile Arg His Ile Pro Arg Arg Ile 325 330 335 ArgGln Gly Leu Glu Arg Ile Leu Leu 340 345 26 amino acids amino acid singlelinear peptide 50 Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln GlnLeu Leu Gly 1 5 10 15 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys 20 25 26amino acids amino acid single linear peptide 51 Arg Ile Leu Ala Val GluArg Tyr Leu Lys Asp Gln Gln Leu Leu Gly 1 5 10 15 Ile Trp Gly Cys SerGly Lys Ile Ile Cys 20 25 26 amino acids amino acid single linearpeptide 52 Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu LeuGly 1 5 10 15 Ile Trp Gly Cys Ser Gly Lys His Ile Cys 20 25 26 aminoacids amino acid single linear peptide 53 Arg Val Leu Ala Val Glu ArgTyr Leu Lys Asp Gln Gln Leu Met Gly 1 5 10 15 Ile Trp Gly Cys Ser GlyLys Leu Ile Cys 20 25 26 amino acids amino acid single linear peptide 54Arg Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly 1 5 1015 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys 20 25 26 amino acids aminoacid single linear peptide 55 Arg Val Leu Ala Val Glu Arg Tyr Leu ArgAsp Gln Gln Leu Leu Gly 1 5 10 15 Ile Trp Gly Cys Ser Gly Arg His IleCys 20 25 26 amino acids amino acid single linear peptide 56 Arg Val LeuAla Val Glu Arg Tyr Leu Gln Asp Gln Arg Leu Leu Gly 1 5 10 15 Met TrpGly Cys Ser Gly Lys His Ile Cys 20 25 26 amino acids amino acid singlelinear peptide 57 Arg Leu Leu Ala Val Glu Arg Tyr Leu Gln Asp Gln GlnIle Leu Gly 1 5 10 15 Leu Trp Gly Cys Ser Gly Lys Ala Val Cys 20 25 26amino acids amino acid single linear peptide 58 Arg Leu Leu Ala Leu GluThr Phe Ile Gln Asn Gln Gln Leu Leu Asn 1 5 10 15 Leu Trp Gly Cys LysAsn Arg Leu Ile Cys 20 25 26 amino acids amino acid single linearpeptide 59 Arg Leu Gln Ala Leu Glu Thr Leu Ile Gln Asn Gln Gln Arg LeuAsn 1 5 10 15 Leu Trp Gly Cys Lys Gly Lys Leu Ile Cys 20 25 26 aminoacids amino acid single linear peptide 60 Arg Leu Leu Ala Leu Glu ThrLeu Leu Gln Asn Gln Gln Leu Leu Ser 1 5 10 15 Leu Trp Gly Cys Lys GlyLys Leu Val Cys 20 25 26 amino acids amino acid single linear peptide 61Arg Val Thr Ala Ile Glu Lys Tyr Leu Gln Asp Gln Ala Arg Leu Asn 1 5 1015 Ser Trp Gly Cys Ala Phe Arg Gln Val Cys 20 25 26 amino acids aminoacid single linear peptide 62 Arg Val Thr Ala Ile Glu Lys Tyr Leu LysAsp Gln Ala Gln Leu Asn 1 5 10 15 Ser Trp Gly Cys Ala Phe Arg Gln ValCys 20 25 2621 base pairs nucleic acid single linear DNA 63 ATGACAGCGATTATGAAAGC AATGGGGAAG AGGAACAGGA AGTTAGGGAT CTGGTGCTTG 60 ATTTTGGCTTTGATAATCCC ATGTTTGAGC TGTAACCAAC TATATGCCAC AGTCTATTCT 120 GGGGTACCTGTATGGGAAGA TGCAAAACCA ACATTGTTCT GTGCTTCAGA TGCTAACTTG 180 ACAAGCACTGAACAGCATAA TATTTGGGCA ACACAAGCCT GTGTTCCCAC AGACCCCAGT 240 CCAAATGAATATGAGCTAAA AAATGTGACA GGTAAATTCA ATATATGGAA AAATTATATA 300 GTAGACCAAATGCACGAAGA CATTATAGAT TTGTGGGACC AGAGTTTAAA ACCTTGTGTT 360 CAAATGACTTTCTTGTGTGT ACAAATGAAT TGTACAGATA TCAAAAATAG TATTAATACC 420 ACAAACAGTCCCTTAAACTC AAACAATACA AAAGAGGTGA AACAGTGTGA CTTTAATGTA 480 ACTACAGTGCTCAAAGACAA ACAGGAGAAA AAACAGGCTC TATTCTATGT GACAGATTTG 540 GTTAAGATTAACGCCACATC AAATGAAACA ATGTATAGAT TAATTAATTG TAACTCCACA 600 ACCATCAGGCAGGCCTGTCC AAAGGTATCT TTTGAGCCCA TTCCCATACA CTATTGTGCT 660 CCAGCGGGATGTGCCATCTT TAAGTGTAAT GAAACAGGAT TTAATGGAAC AGGTCTCTGT 720 AAAAACGTTACAGTAGTTAC TTGTACACAT GGCATCAAAC CAACAGTAAG TACCCAACTA 780 ATACTAAATGGGACACTCTC TAAAGGAAAT ATAACAATCA TGGGAAAGAA TATTTCAGAC 840 AGTGGGGAGAACATCCTAAT AACCCTAAAT ACTAATATAA CAATAGCATG TGAGAGACCA 900 GGAAATCAGACAATACAAAA GATAATGGCA GGTCCAATGG CTTGGTACAG CATGGCCCTT 960 AGTAATACAAAGGGGGATAC AAGGGCAGCT TATTGTAATT ATAGTGCCAC TGACTGGAAC 1020 AAAGCCTTAAAAAACATAAC TGAAAGATAT TTAGAACTTG TAGAATATAA TCAAACTGAT 1080 GTTACCATGAAATTCGGTAA TCACAGTGGT GAAGATGCAG AAGTAACAAA TTTCTTTTTT 1140 AACTGTCATGGAGAATTCTT TTATTGTAAC ACAAATCGGC TGTTTAATCA TACCTTTTCC 1200 TGCAAGAAGAATATGACCAA TAACAAGATC AATTGTACTA ATATTAGCAA TAATAGCAAT 1260 GGCACTCAGGCAATACCTTG CAGGTTGAGA CAAGTAGTAA GGGACTGGAT TCGGGACTTT 1320 ATGCACCTCCCATCCCAGGA AACCTAGTAT GCAGGTCAAA CATAACTGGA ATGATTCTAC 1380 AATTGGACACGCCATGGAAT AAAACACATC CTAACAGCAC CACCCTTAGA CCAGGAGGGG 1440 GAGATATGAAAGATATATGG AGAACTCAAT TGTTCAAATA TAAAGTAGTA AGAGTAAAAC 1500 CTTTTAGTGTAGCACCAACA AAAATTGCAA GGCCAACTAT AGGAACTAGA TCTCATAGAG 1560 AGAAAAGAGCAGCAGGTTTG GCAATGCTAT TCTTGGGGAT TCTAAGTGCA GCAGGAAGCA 1620 CTATGGGCGCAGCGGCAACA GCGCTGACGG TACGGACCCA GCATCTGATA AAGGGTATAG 1680 TGCAACAGCAGGATAACCTG CTAAGAGCAA TACAGGCCCA GCAACACTTG CTGAGGCCAT 1740 CTGTATGGGGTATTAGACAA CTCCGAGCTC GCCTGCTAGC CTTAGAAACC TTTATACAGA 1800 ATCAGCAACTCCTTAACCTG TGGGGCTGCA AGAATAGACT AATCTGCTAC ACATCAGTAA 1860 AGTGGAATAAAACATGGGGA GGAGATAATG AATCAATTTG GGATGAGTTA ACATGGCAGC 1920 AGTGGGATCAACAGATAAAC AACGTAAGCT CCTTCATATA TGAAAAAATA CAAGAGGCAC 1980 AAGAACAACAGGAGAAAAAT GAGAAAGAAT TGCTGGAGTT AGATGAATGG GCCTCTATTT 2040 GGAATTGGCTTGACATAACT AAATGGTTGT GGTATATAAA AATAGCTATA ATCATAGTAG 2100 GAGCACTAATAGGTGTAAGA GTAGTTATGA TAGTACTTAA TCTAGTAAAG AACATTAGGC 2160 AGGGATATCAACCCCTCTCG TTACAGATCC CCATCCAACA ACAAGCGGAA GTAGGAACGC 2220 CAGGAGGAACAGGAGAAGGA GGTGGAGACG AAGACAGGCG CAGGTGGACT CCATTGCCGC 2280 AAGGGTTCTTGCATCTGTTG TACACGGACC TCAGGACAAT AATCTTGTGG ATTTACCACC 2340 TCTTGAGCAACTTAGCCTCA GAGATCCAGA AGTTGATCAG ACACCTGGGA CTTGGACTAT 2400 GGATCATAGGGCAGAGGACA ATTGAAGCTT GCAGACTCTT TAAAGCTATA ATACAATACT 2460 GGCTACAAGAATTGCAAACT AGTGCTACAA ATCTACTAGA TACTGTTGCA GTGGCAGTTG 2520 CTAATTGGACTGACAGCACA ATCTTAGGCA TACAAAGCAT AGGGAGAGGG ATTCTTAACA 2580 TACCAAGAAGGATTAGACAG GGCCTTGAAC GACTCCTGTT A 2621 453 base pairs nucleic acidsingle linear DNA 64 GCAGAGACAG GACAGGAAAC TGCCTACTTC CTGTTAAAATTAGCAGCAAG ATGGCCTATT 60 AAAATACTAC ATACAGACAA TGGGCCTAAC TTTACAAGTGCAGCCATGAA AGCTGCATGT 120 TGGTGGACAA ACATACAACA TGAGTTTGGA ATACCATACAATCCACAAAG TCAAGGAGTA 180 GTAGAAGCCA TGAACAAGGA ATTAAAATCA ATCATACAGGTGAGGGACCA AGCAGAGCAC 240 TTAAGGACAG CAGTACAAAT GGCAGTATTT GTTCACAATTTTAAAAGAAA AGGGGGGATT 300 GGGGGGTACA CTGCAGGAGA GAGATTAATA GACATATTAGCATCACAAAT ACAAACAACA 360 GAACTACAAA AACAAATTTT AAAAATTCAA AATTTTCGGGTCTATTACAG AGACAGCAGA 420 GACCCTATTT GGAAAGGACC GGCACAGCTC CTG 453 170amino acids amino acid single linear peptide 65 Gln Gly Gln Met Val HisGln Ala Leu Ser Pro Arg Thr Leu Asn Ala 1 5 10 15 Trp Val Lys Ala ValGlu Glu Lys Ala Phe Asn Pro Glu Ile Ile Pro 20 25 30 Met Phe Met Ala LeuSer Glu Gly Ala Val Pro Tyr Asp Ile Asn Val 35 40 45 Met Leu Asn Ala IleGly Gly His Gln Gly Ala Leu Gln Val Leu Lys 50 55 60 Glu Val Ile Asn AspGlu Ala Ala Asp Trp Asp Arg Ala His Pro Gln 65 70 75 80 Gln Ala Gly ProLeu Pro Pro Gly Gln Ile Arg Glu Pro Thr Gly Ser 85 90 95 Asp Ile Ala GlyThr Thr Ser Thr Gln Gln Glu Gln Ile Leu Trp Thr 100 105 110 Thr Arg AlaGly Asn Pro Ile Pro Val Gly Asp Ile Tyr Arg Lys Trp 115 120 125 Ile ValLeu Gly Leu Asn Lys Met Val Lys Met Tyr Ser Pro Val Ser 130 135 140 IleLeu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val 145 150 155160 Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu 165 170 170 amino acidsamino acid single linear peptide 66 Gln Gly Gln Met Val His Gln Ala IleSer Pro Arg Thr Leu Asn Ala 1 5 10 15 Trp Val Lys Ala Val Glu Glu LysAla Phe Asn Pro Glu Ile Ile Pro 20 25 30 Met Phe Met Ala Leu Ser Glu GlyAla Ile Ser Tyr Asp Ile Asn Thr 35 40 45 Met Leu Asn Ala Ile Gly Gly HisGln Gly Ala Leu Gln Val Leu Lys 50 55 60 Glu Val Ile Asn Glu Glu Ala ValGlu Trp Asp Arg Thr His Pro Pro 65 70 75 80 Pro Val Gly Pro Leu Pro ProGly Gln Ile Arg Glu Pro Thr Gly Ser 85 90 95 Asp Ile Ala Gly Thr Thr SerThr Gln Gln Glu Gln Ile His Trp Thr 100 105 110 Thr Arg Pro Asn Gln ProIle Pro Val Gly Asp Ile Tyr Arg Lys Trp 115 120 125 Ile Val Leu Gly LeuAsn Lys Met Val Lys Met Tyr Ser Pro Val Ser 130 135 140 Ile Leu Asp IleLys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val 145 150 155 160 Asp ArgPhe Tyr Lys Thr Leu Arg Ala Glu 165 170 170 amino acids amino acidsingle linear peptide 67 Gln Gly Gln Met Val His Gln Ala Ile Ser Pro ArgThr Leu Asn Ala 1 5 10 15 Trp Val Lys Ala Val Glu Glu Lys Ala Phe AsnPro Glu Ile Ile Pro 20 25 30 Met Phe Met Ala Leu Ser Glu Gly Ala Val ProTyr Asp Ile Asn Thr 35 40 45 Met Leu Asn Ala Ile Gly Gly His Gln Gly AlaLeu Gln Val Leu Lys 50 55 60 Glu Val Ile Asn Glu Glu Ala Ala Glu Trp AspArg Thr His Pro Pro 65 70 75 80 Ala Met Gly Pro Leu Pro Pro Gly Gln IleArg Glu Pro Thr Gly Ser 85 90 95 Asp Ile Ala Gly Thr Thr Ser Thr Gln GlnGlu Gln Ile Ile Trp Thr 100 105 110 Thr Arg Gly Ala Asn Ser Ile Pro ValGly Asp Ile Tyr Arg Lys Trp 115 120 125 Ile Val Leu Gly Leu Asn Lys MetVal Lys Met Tyr Ser Pro Val Ser 130 135 140 Ile Leu Asp Ile Arg Gln GlyPro Lys Glu Pro Phe Arg Asp Tyr Val 145 150 155 160 Asp Arg Phe Tyr LysThr Leu Arg Ala Glu 165 170 169 amino acids amino acid single linearpeptide 68 Gln Gly Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu AsnAla 1 5 10 15 Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu ValIle Pro 20 25 30 Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp LeuAsn Thr 35 40 45 Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln MetLeu Lys 50 55 60 Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Val HisPro Val 65 70 75 80 His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu ProArg Gly Ser 85 90 95 Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln IleGly Trp Met 100 105 110 Thr Asn Asn Pro Pro Ile Pro Val Gly Glu Ile TyrLys Arg Trp Ile 115 120 125 Ile Leu Gly Leu Asn Lys Ile Val Arg Met TyrSer Pro Thr Ser Ile 130 135 140 Leu Asp Ile Arg Gln Gly Pro Lys Glu ProPhe Arg Asp Tyr Val Asp 145 150 155 160 Arg Phe Tyr Lys Thr Leu Arg AlaGlu 165 169 amino acids amino acid single linear peptide 69 Gln Gly GlnMet Ile His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala 1 5 10 15 Trp ValLys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro 20 25 30 Met PheSer Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Met 35 40 45 Met LeuAsn Ile Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys 50 55 60 Asp ThrIle Asn Glu Glu Ala Ala Asp Trp Asp Arg Val His Pro Val 65 70 75 80 HisAla Gly Pro Ile Pro Pro Gly Gln Met Arg Glu Pro Arg Gly Ser 85 90 95 AspIle Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp Met 100 105 110Thr Ser Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile 115 120125 Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile 130135 140 Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp145 150 155 160 Arg Phe Phe Lys Thr Leu Arg Ala Glu 165 169 amino acidsamino acid single linear peptide 70 Gln Gly Gln Met Val His Gln Ala IleSer Pro Arg Thr Leu Asn Ala 1 5 10 15 Trp Val Lys Val Val Glu Glu LysAla Phe Ser Pro Glu Val Ile Pro 20 25 30 Met Phe Ser Ala Leu Ser Glu GlyAla Leu Pro Gln Asp Val Asn Thr 35 40 45 Met Leu Asn Ala Val Gly Gly HisGln Gly Ala Met Gln Val Leu Lys 50 55 60 Glu Val Ile Asn Glu Glu Ala AlaGlu Trp Asp Arg Leu His Pro Thr 65 70 75 80 His Ala Gly Pro Ile Ala ProGly Gln Leu Arg Glu Pro Arg Gly Ser 85 90 95 Asp Ile Ala Gly Thr Thr SerThr Leu Gln Glu Gln Ile Gly Trp Thr 100 105 110 Thr Ala Asn Pro Pro IlePro Val Gly Asp Val Tyr Arg Arg Trp Val 115 120 125 Ile Leu Gly Leu AsnLys Val Val Arg Met Tyr Cys Pro Val Ser Ile 130 135 140 Leu Asp Ile ArgGln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp 145 150 155 160 Arg PheTyr Lys Thr Leu Arg Ala Glu 165 35 amino acids amino acid single linearpeptide 71 Cys Thr Arg Pro Tyr Lys Asn Thr Arg Gln Arg Thr Gly Ile GlyPro 1 5 10 15 Gly Gln Ala Leu Tyr Thr Thr His Arg Ile Ile Gly Asp IleArg Gln 20 25 30 Ala His Cys 35 37 amino acids amino acid single linearpeptide 72 Arg Val Leu Ala Val Glu Arg Tyr Leu Gln Asp Gln Gln Leu LeuGly 1 5 10 15 Ile Trp Gly Cys Ser Gly Lys His Ile Cys Thr Thr Thr ValPro Trp 20 25 30 Asn Ser Ser Trp Ser 35 36 amino acids amino acid singlelinear peptide 73 Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile ArgIle Gln Arg 1 5 10 15 Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys IleGly Asn Met Arg 20 25 30 Gln Ala His Cys 35 37 amino acids amino acidsingle linear peptide 74 Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp GlnGln Leu Leu Gly 1 5 10 15 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys ThrThr Ala Val Pro Trp 20 25 30 Asn Ala Ser Trp Ser 35 37 amino acids aminoacid single linear peptide 75 Cys Thr Arg Pro Asn Asn Asn Thr Arg AsnArg Ile Ser Ile Gln Arg 1 5 10 15 Gly Pro Gly Arg Ala His Val Thr ThrLys Gln Ile Ile Gly Asp Ile 20 25 30 Arg Gln Ala His Cys 35 37 aminoacids amino acid single linear peptide 76 Arg Val Leu Ala Val Glu ArgTyr Leu Lys Asp Gln Gln Leu Leu Gle 1 5 10 15 Ile Trp Gly Cys Ser GlyLys Leu Ile Cys Thr Thr Thr Val Pro Trp 20 25 30 Asn Ala Ser Trp Ser 3534 amino acids amino acid single linear peptide 77 Cys Ala Arg Pro TyrGln Asn Thr Arg Gln Arg Thr Pro Ile Gly Leu 1 5 10 15 Gly Gln Ser LeuTyr Thr Thr Arg Ser Arg Ser Ile Ile Gly Gln Ala 20 25 30 His Cys 37amino acids amino acid single linear peptide 78 Arg Ile Leu Ala Val GluArg Tyr Leu Lys Asp Gln Gln Leu Leu Gly 1 5 10 15 Ile Trp Gly Cys SerGly Lys His Ile Cys Thr Thr Asn Val Pro Trp 20 25 30 Asn Ser Ser Trp Ser35 33 amino acids amino acid single linear peptide 79 Cys Thr Arg ProGly Asn Asn Thr Arg Arg Gly Ile His Phe Gly Pro 1 5 10 15 Gly Gln AlaLeu Tyr Thr Thr Gly Val Gly Asp Ile Arg Arg Ala Tyr 20 25 30 Cys 37amino acids amino acid single linear peptide 80 Arg Val Leu Ala Val GluArg Tyr Leu Gln Asp Gln Arg Leu Leu Gly 1 5 10 15 Met Trp Gly Cys SerGly Lys His Ile Cys Thr Thr Phe Val Pro Trp 20 25 30 Asn Ser Ser Trp Ser35 36 amino acids amino acid single linear peptide 81 Cys Ser Arg ProTyr Asn Thr Arg Lys Asn Ile Arg Arg Tyr Ser Ile 1 5 10 15 Gly Ser GlyGln Ala Phe Tyr Val Thr Gly Lys Ile Gly Asp Ile Arg 20 25 30 Gln Ala HisCys 35 37 amino acids amino acid single linear peptide 82 Arg Val LeuAla Val Glu Arg Tyr Leu Gln Asp Gln Gln Leu Leu Gly 1 5 10 15 Ile TrpGly Cys Ser Gly Lys Leu Ile Cys Thr Thr Thr Val Pro Trp 20 25 30 Asn SerSer Trp Ser 35 35 amino acids amino acid single linear peptide 83 CysHis Arg Pro Gly Asn Asn Thr Arg Gly Glu Val Gln Ile Gly Pro 1 5 10 15Gly Met Thr Phe Tyr Asn Ile Glu Asn Val Val Gly Asp Thr Arg Ser 20 25 30Ala Tyr Cys 35 37 amino acids amino acid single linear peptide 84 ArgLeu Leu Ala Val Glu Arg Tyr Leu Gln Asp Gln Gln Ile Leu Gly 1 5 10 15Leu Trp Gly Cys Ser Gly Lys Ala Val Cys Tyr Thr Thr Val Pro Trp 20 25 30Asn Asn Ser Trp Pro 35 36 amino acids amino acid single linear peptide85 Cys Glu Arg Pro Gln Ile Asp Ile Gln Glu Met Arg Ile Gly Pro Met 1 510 15 Ala Trp Tyr Ser Met Gly Ile Gly Gly Thr Ala Gly Asn Ser Ser Arg 2025 30 Gln Ala Tyr Cys 35 37 amino acids amino acid single linear peptide86 Arg Leu Leu Ala Leu Glu Thr Leu Leu Gln Asn Gln Gln Leu Leu Ser 1 510 15 Leu Trp Gly Cys Lys Gly Lys Leu Val Cys Tyr Thr Ser Val Lys Trp 2025 30 Asn Arg Thr Trp Ile 35 36 amino acids amino acid single linearpeptide 87 Cys Ile Arg Glu Gly Ile Ala Glu Val Gln Asp Ile Tyr Thr GlyPro 1 5 10 15 Met Arg Trp Arg Ser Met Thr Leu Ile Arg Ser Asn Asn ThrSer Arg 20 25 30 Val Ala Tyr Cys 35 37 amino acids amino acid singlelinear peptide 88 Arg Leu Gln Ala Leu Glu Thr Leu Ile Gln Asn Gln GlnArg Leu Asn 1 5 10 15 Leu Trp Gly Cys Lys Gly Lys Leu Ile Cys Tyr ThrSer Val Lys Trp 20 25 30 Asn Arg Thr Trp Ile 35 36 amino acids aminoacid single linear peptide 89 Cys Glu Arg Pro Gly Asn Gln Thr Ile GlnLys Ile Met Ala Gly Pro 1 5 10 15 Met Ala Trp Tyr Ser Met Ala Leu SerAsn Thr Lys Gly Asp Thr Arg 20 25 30 Ala Ala Tyr Cys 35 37 amino acidsamino acid single linear peptide 90 Arg Leu Leu Ala Leu Glu Thr Phe IleGln Asn Gln Gln Leu Leu Asn 1 5 10 15 Leu Trp Gly Cys Lys Asn Arg LeuIle Cys Tyr Thr Ser Val Lys Trp 20 25 30 Asn Lys Thr Trp Gly 35 40 aminoacids amino acid single linear peptide 91 Cys Xaa Arg Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa 1 5 10 15 Gly Pro Met Xaa Trp XaaSer Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Ser Arg Xaa AlaXaa Cys 35 40 37 amino acids amino acid single linear peptide 92 Arg LeuXaa Ala Leu Glu Thr Xaa Xaa Gln Asn Gln Gln Xaa Leu Xaa 1 5 10 15 LeuTrp Gly Cys Xaa Xaa Xaa Xaa Xaa Cys Tyr Thr Ser Val Xaa Trp 20 25 30 AsnXaa Thr Trp Xaa 35 38 amino acids amino acid single linear peptide 93Cys Val Arg Pro Gly Asn Asn Ser Val Lys Glu Ile Lys Ile Gly Pro 1 5 1015 Met Ala Trp Tyr Ser Met Gln Ile Glu Arg Glu Gly Lys Gly Ala Asn 20 2530 Ser Arg Thr Ala Phe Cys 35 37 amino acids amino acid single linearpeptide 94 Arg Leu Leu Ala Leu Glu Thr Leu Met Gln Asn Gln Gln Leu LeuAsn 1 5 10 15 Leu Trp Gly Cys Arg Gly Lys Ala Ile Cys Tyr Thr Ser ValGln Trp 20 25 30 Asn Glu Thr Trp Gly 35 513 base pairs nucleic acidsingle linear DNA 95 CAGGGACAAA TGGTACATCA GGCCATCTCC CCCAGAACTTTATATGTATG GGTAAAGGCA 60 GTAGAAGAAA AGGCCTTTAA CCCTGAAATT ATCCCTATGTTTATGGCACT ATCAGAAGGA 120 GCTGTTCCCT ATGATATCAA TGTTATGCTA AATGCCATAGGAGGACACCA AGGGGCTTTA 180 CAAGTATTAA AAGAAGTAAT CAATGATGAA GCAGCAGACTGGGATAGAGC TCACCCACAA 240 CAGGCAGGGC CGTTACCACC AGGGCAGATA AGGGAACCAACAGGAAGTGA CATTGCTGGA 300 ACAACTAGCA CACAGCAAGA GCAAATTCTC TGGACTACTAGGGCAGGTAA CCCTATCCCA 360 GTTGGAGACA TCTATAGGAA ATGGATAGTG TTGGGTCTAAACAAAATGGT AAAAATGTAT 420 AGTCCAGTGA GCATCTTAGA TATTAGGCAG GGACCAAAAGAACCATTTAG AGATTATGTA 480 GACAGGTTCT ACAAAACATT AAGAGCTGAG CAG 513 171amino acids amino acid single linear peptide 96 Gln Gly Gln Met Val HisGln Ala Leu Ser Pro Arg Thr Leu Asn Ala 1 5 10 15 Trp Val Lys Ala ValGlu Glu Lys Ala Phe Asn Pro Glu Ile Ile Pro 20 25 30 Met Phe Met Ala LeuSer Glu Gly Ala Val Pro Tyr Asp Ile Asn Val 35 40 45 Met Leu Asn Ala IleGly Gly His Gln Gly Ala Leu Gln Val Leu Lys 50 55 60 Glu Val Ile Asn AspGlu Ala Ala Asp Trp Asp Arg Ala His Pro Gln 65 70 75 80 Gln Ala Gly ProLeu Pro Pro Gly Gln Ile Arg Glu Pro Thr Gly Ser 85 90 95 Asp Ile Ala GlyThr Thr Ser Thr Gln Gln Glu Gln Ile Leu Trp Thr 100 105 110 Thr Arg AlaGly Asn Pro Ile Pro Val Gly Asp Ile Tyr Arg Lys Trp 115 120 125 Ile ValLeu Gly Leu Asn Lys Met Val Lys Met Tyr Ser Pro Val Ser 130 135 140 IleLeu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val 145 150 155160 Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln 165 170 525 base pairsnucleic acid single linear DNA 97 ATTCCAATAC ACTATTGTGC TCCAGCAGGATATGCTATCT TTAAATGCAA CAACGAGGAG 60 TTTACTGGAA AAGGCCCATG TAACAACATTTCAGTAGTTA CCTGTACACA GGGTATCAAG 120 CCAACAGTAA GCACTCATCT AATATTCAATGGGACAATCT CTGAAAGAAA AATAAGAATT 180 ATGGGAAAGA ACATCTCGAG CAACTCAGGTAATATCCTAG TGACCCTAAA TTCTACTATA 240 AACATGACCT GTGTGAGGCC AGGAAATAATTCAGTACAGG AGATAAAAAT AGGTCCAATG 300 GCTTGGTACA GTATGCAAAT TGAGCGAGAGGGAAAAGGAG CAAATTCAAG AACAGCTTTT 360 TGTACCTATA ATGCCACGGA CTGGAGAAAAACCTTGCAAG GGATAGCTGA AAGGTATTTA 420 GAACTTGTAA ATAAAACAAG TCCGACTGAAATAATGTTCA ATAAAAGCAA TGGTGGAGAT 480 GCAGAAATAA CCCGTTTGCA TTTTAACTGTCATGGAGAAT TCTTT 525 175 amino acids amino acid single linear peptide 98Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Tyr Ala Ile Phe Lys Cys 1 5 1015 Asn Asn Glu Glu Phe Thr Gly Lys Gly Pro Cys Asn Asn Ile Ser Val 20 2530 Val Thr Cys Thr Gln Gly Ile Lys Pro Thr Val Ser Thr His Leu Ile 35 4045 Phe Asn Gly Thr Ile Ser Glu Arg Lys Ile Arg Ile Met Gly Lys Asn 50 5560 Ile Ser Ser Asn Ser Gly Asn Ile Leu Val Thr Leu Asn Ser Thr Ile 65 7075 80 Asn Met Thr Cys Val Arg Pro Gly Asn Asn Ser Val Gln Glu Ile Lys 8590 95 Ile Gly Pro Met Ala Trp Tyr Ser Met Gln Ile Glu Arg Glu Gly Lys100 105 110 Gly Ala Asn Ser Arg Thr Ala Phe Cys Thr Tyr Asn Ala Thr AspTrp 115 120 125 Arg Lys Thr Leu Gln Gly Ile Ala Glu Arg Tyr Leu Glu LeuVal Asn 130 135 140 Lys Thr Ser Pro Thr Glu Ile Met Phe Asn Lys Ser AsnGly Gly Asp 145 150 155 160 Ala Glu Ile Thr Arg Leu His Phe Asn Ser CysGly Glu Phe Phe 165 170 175 312 base pairs nucleic acid single linearDNA 99 ATAGTGCAAC AGCAGGACAA CCTGCTGAGA GCAATACAGG CCCAGCAACA TCTGCTGAGG60 TTATCTGTAT GGGGTATTAG ACAACTCCGA GCTCGCCTGC TAGCCTTAGA AACCCTTATG 120CAGAATCAGC AACTCCTAAA CCTGTGGGGT TGTAGAGGAA AAGCAATCTG CTACACATCA 180GTACAATGGA ATGAAACATG GGGAGGAAAT GACTCAATTT GGGACAGGTT AACATGGCAG 240CAATGGGATC AACAGATAGC CAATGTAAGC TCTTTTATAT ATGACAAAAT ACAAGAAGCA 300CAAGAACAAC AA 312 104 amino acids amino acid single linear peptide 100Ile Val Gln Gln Gln Asp Asn Leu Leu Arg Ala Ile Gln Ala Gln Gln 1 5 1015 His Leu Leu Arg Leu Ser Val Trp Gly Ile Arg Gln Leu Arg Ala Arg 20 2530 Leu Leu Ala Leu Glu Thr Leu Met Gln Asn Gln Gln Leu Leu Asn Leu 35 4045 Trp Gly Cys Arg Gly Lys Ala Ile Cys Tyr Thr Ser Val Gln Trp Asn 50 5560 Glu Thr Trp Gly Gly Asn Asp Ser Ile Trp Asp Arg Leu Thr Trp Gln 65 7075 80 Gln Trp Asp Gln Gln Ile Ala Asn Val Ser Ser Phe Ile Tyr Asp Lys 8590 95 Ile Gln Glu Ala Gln Glu Gln Gln 100 35 amino acids amino acidsingle linear peptide 101 Arg Leu Leu Ala Leu Glu Thr Phe Ile Gln AsnGln Gln Leu Leu Asn 1 5 10 15 Leu Trp Gly Cys Lys Asn Arg Leu Ile CysTyr Thr Ser Val Lys Trp 20 25 30 Asn Lys Thr 35 877 amino acids aminoacid single linear protein 102 Met Thr Ala Ile Met Lys Ala Met Gly LysArg Asn Arg Lys Leu Gly 1 5 10 15 Ile Trp Cys Leu Ile Leu Ala Leu IleIle Pro Cys Leu Ser Cys Asn 20 25 30 Gln Leu Tyr Ala Thr Val Tyr Ser GlyVal Pro Val Trp Glu Asp Ala 35 40 45 Lys Pro Thr Leu Phe Cys Ala Ser AspAla Asn Leu Thr Ser Thr Glu 50 55 60 Gln His Asn Ile Trp Ala Thr Gln AlaCys Val Pro Thr Asp Pro Ser 65 70 75 80 Pro Asn Glu Tyr Glu Leu Lys AsnVal Thr Gly Lys Phe Asn Ile Trp 85 90 95 Lys Asn Tyr Ile Val Asp Gln MetHis Glu Asp Ile Ile Asp Leu Trp 100 105 110 Asp Gln Ser Leu Lys Pro CysVal Gln Met Thr Phe Leu Cys Val Gln 115 120 125 Met Asn Cys Thr Asp IleLys Asn Ser Ile Asn Thr Thr Asn Ser Pro 130 135 140 Leu Asn Ser Asn AsnThr Lys Glu Val Lys Gln Cys Asp Phe Asn Val 145 150 155 160 Thr Thr ValLeu Lys Asp Lys Gln Glu Lys Lys Gln Ala Leu Phe Tyr 165 170 175 Val ThrAsp Leu Val Lys Ile Asn Ala Thr Ser Asn Glu Thr Met Tyr 180 185 190 ArgLeu Ile Asn Cys Asn Ser Thr Thr Ile Arg Gln Ala Cys Pro Lys 195 200 205Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Cys 210 215220 Ala Ile Phe Lys Cys Asn Glu Thr Gly Phe Asn Gly Thr Gly Leu Cys 225230 235 240 Lys Asn Val Thr Val Val Thr Cys Thr His Gly Ile Lys Pro ThrVal 245 250 255 Ser Thr Gln Leu Ile Leu Asn Gly Thr Leu Ser Lys Gly AsnIle Thr 260 265 270 Ile Met Gly Lys Asn Ile Ser Asp Ser Gly Glu Asn IleLeu Ile Thr 275 280 285 Leu Asn Thr Asn Ile Thr Ile Ala Cys Glu Arg ProGly Asn Gln Thr 290 295 300 Ile Gln Lys Ile Met Ala Gly Pro Met Ala TrpTyr Ser Met Ala Leu 305 310 315 320 Ser Asn Thr Lys Gly Asp Thr Arg AlaAla Tyr Cys Asn Tyr Ser Ala 325 330 335 Thr Asp Trp Asn Lys Ala Leu LysAsn Ile Thr Glu Arg Tyr Leu Glu 340 345 350 Leu Val Glu Tyr Asn Gln ThrAsp Val Thr Met Lys Phe Gly Asn His 355 360 365 Ser Gly Glu Asp Ala GluVal Thr Asn Phe Phe Phe Asn Cys His Gly 370 375 380 Glu Phe Phe Tyr CysAsn Thr Asn Arg Leu Phe Asn His Thr Phe Ser 385 390 395 400 Cys Lys LysAsn Met Thr Asn Asn Lys Ile Asn Cys Thr Asn Ile Ser 405 410 415 Asn AsnSer Asn Gly Thr Gln Ala Ile Pro Cys Arg Leu Arg Gln Val 420 425 430 ValArg Asp Trp Met Arg Gly Gly Ser Gly Leu Tyr Ala Pro Pro Ile 435 440 445Pro Gly Asn Leu Val Cys Arg Ser Asn Ile Thr Gly Met Ile Leu Gln 450 455460 Leu Asp Thr Pro Trp Asn Lys Thr His Pro Asn Ser Thr Thr Leu Arg 465470 475 480 Pro Gly Gly Gly Asp Met Lys Asp Ile Trp Arg Thr Gln Leu PheLys 485 490 495 Tyr Lys Val Val Arg Val Lys Pro Phe Ser Val Ala Pro ThrLys Ile 500 505 510 Ala Arg Pro Thr Ile Gly Thr Arg Ser His Arg Glu LysArg Ala Ala 515 520 525 Gly Leu Ala Met Leu Phe Leu Gly Ile Leu Ser AlaAla Gly Ser Thr 530 535 540 Met Gly Ala Ala Ala Thr Ala Leu Thr Val ArgThr Gln His Leu Ile 545 550 555 560 Lys Gly Ile Val Gln Gln Gln Asp AsnLeu Leu Arg Ala Ile Gln Ala 565 570 575 Gln Gln His Leu Leu Arg Pro SerVal Trp Gly Ile Arg Gln Leu Arg 580 585 590 Ala Arg Leu Leu Ala Leu GluThr Phe Ile Gln Asn Gln Gln Leu Leu 595 600 605 Asn Leu Trp Gly Cys LysAsn Arg Leu Ile Cys Tyr Thr Ser Val Lys 610 615 620 Trp Asn Lys Thr TrpGly Gly Asp Asn Glu Ser Ile Trp Asp Glu Leu 625 630 635 640 Thr Trp GlnGln Trp Asp Gln Gln Ile Asn Asn Val Ser Ser Phe Ile 645 650 655 Tyr GluLys Ile Gln Glu Ala Gln Glu Gln Gln Glu Lys Asn Glu Lys 660 665 670 GluLeu Leu Glu Leu Asp Glu Trp Ala Ser Ile Trp Asn Trp Leu Asp 675 680 685Ile Thr Lys Trp Leu Trp Tyr Ile Lys Ile Ala Ile Ile Ile Val Gly 690 695700 Ala Leu Ile Gly Val Arg Val Val Met Ile Val Leu Asn Leu Val Lys 705710 715 720 Asn Ile Arg Gln Gly Tyr Gln Pro Leu Ser Leu Gln Ile Pro IleGln 725 730 735 Gln Gln Ala Glu Val Gly Thr Pro Gly Gly Thr Gly Glu GlyGly Gly 740 745 750 Asp Glu Asp Arg Arg Arg Trp Thr Pro Leu Pro Gln GlyPhe Leu His 755 760 765 Leu Leu Tyr Thr Asp Leu Arg Thr Ile Ile Leu TrpIle Tyr His Leu 770 775 780 Leu Ser Asn Leu Ala Ser Glu Ile Gln Lys LeuIle Arg His Leu Gly 785 790 795 800 Leu Gly Leu Trp Ile Ile Gly Gln ArgThr Ile Glu Ala Cys Arg Leu 805 810 815 Phe Lys Ala Ile Ile Gln Tyr TrpLeu Gln Glu Leu Gln Thr Ser Ala 820 825 830 Thr Asn Leu Leu Asp Thr ValAla Val Ala Val Ala Asn Trp Thr Asp 835 840 845 Ser Thr Ile Leu Gly IleGln Ser Ile Gly Arg Gly Ile Leu Asn Ile 850 855 860 Pro Arg Arg Ile ArgGln Gly Leu Glu Arg Leu Leu Leu 865 870 875 861 amino acids amino acidsingle linear protein 103 Met Arg Val Lys Glu Lys Tyr Gln His Leu TrpArg Trp Gly Trp Lys 1 5 10 15 Trp Gly Thr Met Leu Leu Gly Ile Leu MetIle Cys Ser Ala Thr Glu 20 25 30 Lys Leu Trp Val Thr Val Tyr Tyr Gly ValPro Val Trp Lys Glu Ala 35 40 45 Thr Thr Thr Leu Phe Cys Ala Ser Asp AlaLys Ala Tyr Asp Thr Glu 50 55 60 Val His Asn Val Trp Ala Thr His Ala CysVal Pro Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Val Asn ValThr Glu Asn Phe Asn Met Trp 85 90 95 Lys Asn Asp Met Val Glu Gln Met HisGlu Asp Ile Ile Ser Leu Trp 100 105 110 Asp Gln Ser Leu Lys Pro Cys ValLys Leu Thr Pro Leu Cys Val Ser 115 120 125 Leu Lys Cys Thr Asp Leu GlyAsn Ala Thr Asn Thr Asn Ser Ser Asn 130 135 140 Thr Asn Ser Ser Ser GlyGlu Met Met Met Glu Lys Gly Glu Ile Lys 145 150 155 160 Asn Cys Ser PheAsn Ile Ser Thr Ser Ile Arg Gly Lys Val Gln Lys 165 170 175 Glu Tyr AlaPhe Phe Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp 180 185 190 Thr ThrSer Tyr Thr Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln 195 200 205 AlaCys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala 210 215 220Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly 225 230235 240 Thr Gly Pro Cys Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile245 250 255 Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu AlaGlu 260 265 270 Glu Glu Val Val Ile Arg Ser Ala Asn Phe Thr Asp Asn AlaLys Thr 275 280 285 Ile Ile Val Gln Leu Asn Gln Ser Val Glu Ile Asn CysThr Arg Pro 290 295 300 Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln ArgGly Pro Gly Arg 305 310 315 320 Ala Phe Val Thr Ile Gly Lys Ile Gly AsnMet Arg Gln Ala His Cys 325 330 335 Asn Ile Ser Arg Ala Lys Trp Asn AlaThr Leu Lys Gln Ile Ala Ser 340 345 350 Lys Leu Arg Glu Gln Phe Gly AsnAsn Lys Thr Ile Ile Phe Lys Gln 355 360 365 Ser Ser Gly Gly Asp Pro GluIle Val Thr His Ser Phe Asn Cys Gly 370 375 380 Gly Glu Phe Phe Tyr CysAsn Ser Thr Gln Leu Phe Asn Ser Thr Trp 385 390 395 400 Phe Asn Ser ThrTrp Ser Thr Glu Gly Ser Asn Asn Thr Glu Gly Ser 405 410 415 Asp Thr IleThr Leu Pro Cys Arg Ile Lys Gln Phe Ile Asn Met Trp 420 425 430 Gln GluVal Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile 435 440 445 ArgCys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly 450 455 460Asn Asn Asn Asn Gly Ser Glu Ile Phe Arg Pro Gly Gly Gly Asp Met 465 470475 480 Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile485 490 495 Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val ValGln 500 505 510 Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Leu Phe Leu GlyPhe Leu 515 520 525 Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met ThrLeu Thr Val 530 535 540 Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln GlnGln Asn Asn Leu 545 550 555 560 Leu Arg Ala Ile Glu Ala Gln Gln His LeuLeu Gln Leu Thr Val Trp 565 570 575 Gly Ile Lys Gln Leu Gln Ala Arg IleLeu Ala Val Glu Arg Tyr Leu 580 585 590 Lys Asp Gln Gln Leu Leu Gly IleTrp Gly Cys Ser Gly Lys Leu Ile 595 600 605 Cys Thr Thr Ala Val Pro TrpAsn Ala Ser Trp Ser Asn Lys Ser Leu 610 615 620 Glu Gln Ile Trp Asn AsnMet Thr Trp Met Glu Trp Asp Arg Glu Ile 625 630 635 640 Asn Asn Tyr ThrSer Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn 645 650 655 Gln Gln GluLys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala 660 665 670 Ser LeuTrp Asn Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys 675 680 685 IlePhe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe 690 695 700Ala Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu 705 710715 720 Ser Phe Gln Thr His Leu Pro Thr Pro Arg Gly Pro Asp Arg Pro Glu725 730 735 Gly Ile Glu Glu Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser IleArg 740 745 750 Leu Val Asn Gly Ser Leu Ala Leu Ile Trp Asp Asp Leu ArgSer Leu 755 760 765 Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Leu Leu LeuIle Val Thr 770 775 780 Arg Ile Val Glu Leu Leu Gly Arg Arg Gly Trp GluAla Leu Lys Tyr 785 790 795 800 Trp Trp Asn Leu Leu Gln Tyr Trp Ser GlnGlu Leu Lys Asn Ser Ala 805 810 815 Val Ser Leu Leu Asn Ala Thr Ala IleAla Val Ala Glu Gly Thr Asp 820 825 830 Arg Val Ile Glu Val Val Gln GlyAla Cys Arg Ala Ile Arg His Ile 835 840 845 Pro Arg Arg Ile Arg Gln GlyLeu Glu Arg Ile Leu Leu 850 855 860

1. HIV-1 group (or subgroup) O retroviral protein, or natural orsynthetic peptide or polypeptide comprising at least a part of saidprotein, which is capable of being recognized by antibodies which may beisolated from serum obtained after an infection with an HIV-1 group OVAU strain, or an HIV-1 group (or subgroup) O DUR strain.
 2. Protein,polypeptide or peptide according to claim 1, characterized in that itmay be obtained by expression, in a host cell, of a nucleotide sequence,more particularly DNA and cloned DNA fragments which may be obtainedfrom RNA, from cDNA or from primers which may be used for geneamplification, derived from RNA or from DNA of the HIV-1 group (orsubgroup) O retrovirus, said nucleotide sequence being characterized inthat it comprises the sequence corresponding to Seq ID No. 5 as well asany portion of that sequence or variant of that portion which is capableof hybridizing with the corresponding DNA or RNA of the HIV-1 group (orsubgroup) O virus, and in that said protein comprises the amino acidsequence between residues 1 and 526 of Seq ID No. 6 as well as anypeptide, polypeptide, glycoprotein or variant derived from said sequencehaving an epitope which may be recognized by antibodies induced by theHIV-1_((VAU)) virus.
 3. Protein, polypeptide or peptide according toclaim 1 or 2, characterized in that it may be obtained by expression, ina host cell, of a nucleotide sequence according to claim 1, and in thatsaid protein comprises the amino acid sequence between residues 527 to877 of Seq ID No. 7 as well as any peptide, polypeptide, glycoprotein orvariant derived from said sequence having an epitope which may berecognized by antibodies induced by the HIV-1_((VAU)) virus.
 4. Peptideor polypeptide according to claim 1 to 3, characterized in that itcomprises the sequence CKNRLIC or in particular the sequenceRLLALETFIQNWWLLNLWGCKNRLIC or a variant of that sequence such as thesequence RLWALETLIQNQQRLNLWGCKGKLIC, the sequenceRLLALETLLQNQQLLSLWGCKGKLVC, the sequenceRARLLALETFIQNQQLLNLWGCKNRLICYTSVKWNKT, the sequence CERPGNQKIMAGPMAWYSMALSNTKGDTRAAYC or the sequence GPMAWY.
 5. Synthetic peptide,characterized in that it is a protein fragment according to one ofclaims 1 to 4, in that it is obtained from the sequence SEQ ID No. 6 orfrom the sequence SEQ ID No. 7 and in that it is recognized byantibodies induced against an HIV-1_((VAU)) retrovirus or variant ofthis fragment capable of being recognized by antibodies induced by anHIV-1_((VAU)) retrovirus.
 6. Protein, polypeptide or peptide accordingto claim 1, characterized in that said protein is a protein of the HIV-1group (or subgroup) O_((DUR)) virus, deposited on Feb. 23, 1995 at theCNCM under the reference I-1542 or a natural or synthetic peptide orpolypeptide comprising at least a part of said protein or a peptidewhose sequence is distinguished from that of the above by substitution,deletion or addition of amino acids, this separate peptide neverthelessretaining the antigenic characteristics of the above one.
 7. Peptideaccording to claim 6, containing at least 4 consecutive amino acidswhose entire consecutive amino acid sequence is contained in the GAGsequence represented in FIG. 8 or in an immuno-logically similar GAGsequence obtained from a variant of the HIV-1 group (or subgroup) O DURvirus, said immunologically similar sequence being recognized byantibodies which also specifically recognize at least one of thesequences AHPQQA, LWTTRAGNP contained in the GAG sequence of FIG.
 8. 8.Peptide according to claim 7, characterized in that it consists of apeptide whose amino acid sequence is contained either in one of thefollowing sequences: SPRTLNAWVKAVEEKAFNPEIIPMFMALSEGA (1)MLNATGGHQGALQVLKEVIN (2) GPLPPGQIREPTGSDIAGTTSTQQEQI (3)IPVGDIYRKWIVLGLNKMVKMYSPVSILDI (4) QGPKEPFRDYVDRFYKTKLAE (5) AHPQQA (5a)LWTTRAGNP (5b)

or in the corresponding immunologically similar sequence, this peptidecontaining at least 4 consecutive amino acids of one of said sequences.9. Peptide according to claim 8, characterized in that it consists of apeptide whose amino acid sequence is contained either in one of thefollowing sequences: SPRTLNAWVK (6) GSDIAGTTST (7) QGPKEPFRDYVDRF (8)

or in the corresponding immunologically similar sequence, this peptidecontaining at least four consecutive amino acids of one of saidsequences.
 10. Peptide according to claim 8, characterized in that itcontains the following amino acid sequence: NPEI (9).
 11. Peptideaccording to claim 8, characterized in that it contains the followingamino acid sequence: AVEEKAFNPEIIPMFM (10).
 12. Peptide according toclaim 6, containing at least 4 consecutive amino acids, whose entiresequence is contained in the sequence of the region of the V3 loop ofgp120 represented in FIG. 9 or in the corresponding immunologicallysimilar sequence, obtained from a variant of the HIV-1 group (orsubgroup) O DUR virus, said immunologically similar sequence beingrecognized by antibodies which also specifically recognize at least oneof the sequences: KEIKI (12) EREGKGAN (13), CVRPGNNSVKEIKI (14),QIEREGKGANSR (15).


13. Peptide according to claim 12, containing: a) either the sequenceCVRPGNNSVKEIKIGPMAWYSMQIEREGKGANSRTAFC (11) or a part of this sequencewhich contains at least 4 amino acids b) or an amino acid sequence whichis separate from the sequence of a) in which one or more amino acids arereplaced with two amino acids, with the proviso that the peptide retainsits reactivity with an antiserum against the above said peptide, c) oran amino acid sequence which is separate from a) or b), in which one ormore amino acids have been deleted or added, with the proviso that thepeptide retains its reactivity with an antiserum against the peptide ofa), d) or the corresponding immunologically similar sequence or part ofsequence.
 14. Peptide according to claim 13, which contains the sequenceKEIKI (12).
 15. Peptide according to claim 13, which contains thesequence EREGKGAN (13).
 16. Peptide according to claim 13 or 14, whichcontains either the amino acid sequence CVRPGNNSVKEIKI (14) or thesequence QIEREGKGANSR (15).
 17. Peptide according to claim 13, whichcomprises the sequence GPMAWYSM (16).
 18. Peptide according to claim 6,containing at least 4 consecutive amino acids, whose entire sequence iscontained in the sequence of the immunodominant region of gp41represented in FIG. 9 or in the corresponding immunologically similarsequence, obtained from a variant of the HIV-1 group (or subgroup) O DURvirus, said immunologically similar sequence being recognized byantibodies which also specifically recognize at least one of thesequences: RLLALETLMQNQQL (17), LNLWGCRGKAICYTSVQWNETWG (18), CRGKAI(19), SVQWN (20), RLLALETLMONQQLLNLWGCRGKAICYTS (21),QNQQLLNLWGCRGKAICYTSVQWN (22).


19. Peptide according to claim 18, containing the sequenceRLLALETLMQNQQL (17) LNLWGCRGKAICYTSVQWNETWG (18) or part of this peptidecontaining: a) either the sequence CRGKAI (19) or the sequence SVQWN(20) in which Q is, where appropriate, replaced by a different aminoacid, which is nevertheless also different from K, or the two sequencesat the same time, b) or an amino acid sequence which is separate fromthe sequence of a) in which one or more amino acids are replaced withtwo amino acids, with the proviso that the peptide retains itsreactivity with an antiserum against the peptide of a), c) or an aminoacid sequence which is separate from a) or b), in which one or moreamino acids have been deleted or added, with the proviso that thepeptide retains its reactivity with an antiserum against the peptide ofa), d) or in the corresponding immunologically similar sequence or partof sequence.
 20. Peptide according to claim 19, characterized in thatits N-terminal sequence which contains at least 8 amino acids is notimmunologically recognized by antibodies formed against the sequenceRILAVERY contained in the immunodominant region of gp41 of the HIV-1-LAIstrain.
 21. Peptide according to claim 19, characterized in that it isnot recognized by antibodies formed against the peptide SGKLIC of theHIV-1-LAI strain.
 22. Peptide according to claim 19, characterized inthat it contains one or the other of the following two sequences:RLLALETLMONQQLLNLWGCRGKAICYTS (21) QNQQLLNLWGCRGKAICYTSVQWN (22).


23. Nucleotide sequence, more particularly DNA and cloned DNA fragmentswhich may be obtained from RNA, from cDNA or from primers which may beused for gene amplification, derived from the RNA or the DNA of theHIV-1 group (or subgroup) O retrovirus, said nucleotide sequence beingcharacterized in that it comprises the sequence corresponding to one ofthe sequences Seq ID No. 5, Seq ID No. 9, Seq ID No. 10 or Seq ID No.11, as well as any portion of this sequence, in particular the sequencescoding for the proteins, polypeptides or peptides of any one of claims 8to 22 or variant of this portion which is capable of hybridizing withthe corresponding DNA or RNA of the HIV-1 group (or subgroup) O virus.24. Nucleotide sequence according to claim 23, characterized in that itis DNA or DNA fragments obtained from RNA, from cDNA or from primers forgene amplification, derived from the RNA or the DNA of the HIV-1_((VAU))or HIV-1_((DUR)) retrovirus, the sequence comprising the sequencecorresponding to Seq ID No. 5 as well as any portion of this sequence orvariant of this portion which is capable of hybridizing with thecorresponding DNA or RNA of the HIV-1_((VAU)) virus, or the sequencecomprising the sequence corresponding to Seq ID No. 9 or Seq ID No. 10or Seq ID No. 11, as well as any portion of this sequence or variant ofthis portion which is capable of hybridizing with the corresponding DNAor RNA of the HIV-1_((DUR)) virus.
 25. Nucleotide sequence according toclaim 23 or claim 7, characterized in that said sequence is chosen fromthe group of sequences corresponding to Seq ID No. 1, Seq ID No. 2, SeqID No. 3 and Seq ID No.
 4. 26. Nucleotide sequence, characterized inthat it comprises the sequence of nucleotides corresponding to SEQ IDNo. 7 and in that it codes for the integrase of an HIV-1 group (orsubgroup) O retrovirus, in particular of an HIV-1_((VAU)) retrovirus, ornucleotide sequence which hybridizes with the sequence containing thesequence SEQ ID No.
 7. 27. Oligonucleotide comprising at least 9nucleotides, as obtained from a nucleotide sequence according to any oneof claims 23 to 26, which is capable of being used as a primer for thegene amplification of an HIV-1 group (or subgroup) O retrovirus. 28.Oligonucleotide according to claim 27, having a sequence consisting ofat least nine consecutive nucleotides of the following nucleotidesequences: ATT CCA ATA CAC TAT TGT GCT CCA-3′ AAA GAA TTC TCC ATG ACTGTT AAA-3′ GGT ATA GTG CAA CAG CAG GAC AAC-3′ AGA GGC CCA TTC ATC TAACTC-3′


29. Oligonucleotide according to claim 28, characterized in that it maybe used during a process of gene amplification of a nucleotide sequencecoding for a peptide according to any one of claims 6 to
 22. 30.Nucleotide sequence which may be used as a probe, characterized in thatit hybridizes under highly stringent hybridization conditions with theDNA produced by gene amplification by means of primers according to anyone of claims 27 to
 29. 31. Composition for the detection of thepresence or absence of an HIV-1 group (or subgroup) O retrovirus, inparticular the HIV-1_((VAU)) and/or HIV-1_((DUR)) retrovirus, in samplesof serum or of other biological liquids or tissue obtained from patientssuspected of being carriers of an HIV-1 group (or subgroup) Oretrovirus, said composition being characterized in that it comprises atleast one probe obtained from a nucleotide sequence derived from thegenome of the HIV-1_((VAU)) virus, particularly an HIV-1_((VAU)) DNAfragment containing the env region or a part of the env region of theHIV-1_((VAU)) virus, of a variant of HIV-1_((VAU)) as defined in any oneof claims 23 to 27, and/or a probe obtained from a nucleotide sequencederived from the genome of the HIV-1_((DUR)) virus, the HIV-1_((DUR))DNA containing the env region or a part of the env region and a part ofthe GAG region of the HIV-1_((DUR)) virus as defined [lacuna] claim 23or
 24. 32. Composition according to claim 12, characterized in that saidcomposition also comprises a probe obtained from a nucleotide sequenceobtained from HIV-1 not belonging to the O subgroup and/or from HIV-2.33. Composition for the detection of the presence or absence of an HIV-1group (or subgroup) O retrovirus, in particular the HIV-1_((VAU))retrovirus and/or the HIV-1 group (or subgroup) O_((DUR)) retrovirus ina biological sample, said composition being characterized in that itcomprises at least two nucleotide sequences according to any one ofclaims 23 to 27, and at least two nucleotide sequences according toclaim 23 or 24, which are respectively derived from the genome of theHIV-1_((VAU)) and HIV-1_((DUR)) viruses, which sequences can be used asprimers for amplification, in particular by PCR, of the DNA and/or theRNA of HIV-1 retrovirus of the O subgroup and in particular ofHIV-1_((VAU)) and HIV-1_((DUR)).
 34. Nucleotide sequence, characterizedin that it is an RNA sequence corresponding to a DNA sequence accordingto any one of claims 23 to
 31. 35. Composition for the in vitrodetection of the presence, in a human biological sample, ofanti-HIV-1_((VAU)) and anti-HIV-1_((DUR)) antibodies, said compositioncomprising at least one antigen comprising a protein, a glycoprotein, apolypeptide or a peptide of the envelope protein of an HIV-1_((VAU))retrovirus as defined in any one of claims 1 to 5 and/or of the sequencecomprising the sequence corresponding to Seq ID No. 9 or Seq ID No. 10or Seq ID No. 11, as well as any portion of this sequence or variant ofthis portion which is capable of hybridizing with the corresponding DNAor RNA of the HIV-1_((DUR)) virus.
 36. Composition according to claim35, characterized in that it also comprises an antigen such as aprotein, a glycoprotein, a polypeptide or a peptide of an HIV-1 virusnot belonging to the subgroup O and/or of an HIV-2 virus or a peptidederived from an HIV-1 virus not belonging to the subgroup O and/or of anHIV-2 virus having an epitope which may be recognized by the antibodiesinduced by the HIV-1 virus not belonging to the subgroup O and/or theHIV-2 virus.
 37. Composition according to claim 36, characterized inthat the proteins and/or glycoproteins of HIV-1 not belonging to thesubgroup O and/or of HIV-2 are gag or pol proteins or peptides thereof.38. Composition according to claim 37, characterized in that theproteins and/or glycoproteins of HIV-1 not belonging to the subgroup Oand/or of HIV-2 are envelope glycoproteins.
 39. Composition according toany one of claims 35 to 38, characterized in that said compositioncomprises a peptide sequence corresponding to the entire region 590-620of the gp41 protein of HIV-1_((VAU)) or a part of this region which isspecific for HIV-1_((VAU)).
 40. Composition according to claim 20,characterized in that said peptide sequence is the sequence -TFIQN-,CKNRLIC or WGCKNR.
 41. Antibody which may recognize a protein, a peptideor a polypeptide derived from said protein according to any one ofclaims 1 to
 22. 42. Process for the in vitro diagnosis of an infectioncaused by the HIV-1_((VAU)) virus and/or by the HIV-1_((DUR)) virus,said process comprising: the placing in contact of a serum or of anotherbiological medium, derived from a patient forming the subject of thediagnosis, with at least one of the envelope proteins or glycoproteinsof the HIV-1_((VAU)) and/or HIV-1_((DUR)) virus or of a peptide orpolypeptide obtained from one of these proteins or glycoproteinsrespectively according to any one of claims 1 to 5 and according to anyone of claims 6 to 22, or a composition according to any one of claims35 to 38, and the detection of an immunological reaction.
 43. Reagentrequired for the Western blot (immunoblot) or ELISA reaction, containingan envelope protein or glycoprotein of the HIV-1_((VAU)) and/orHIV-1_((DUR)) virus or of a peptide or polypeptide obtained from one ofthese proteins or glycoproteins according to any one of claims 1 to 5and according to any one of claims 6 to 22 or a composition according toany one of claims 35 to
 38. 44. Use of a nucleotide sequence accordingto claim 23 or 24 in order to induce in vivo the synthesis of antibodiesdirected against the antigen coded for by said sequence.
 45. Immunogeniccomposition according to any one of claims 35 to 38, which is capable ofinducing antibodies in animals.
 46. Diagnostic kit for the in vitrodetection, on a biological sample, of an infection with an HIV-1subgroup O retrovirus, for example of an HIV-1_((VAU)) and/orHIV-1_((DUR)) retrovirus, characterized in that it comprises: primersaccording to any one of claims 27 to 29 for the gene amplification of anHIV-1 subgroup O retrovirus, reagents required for the geneamplification reaction.
 47. Kit for the in vitro detection, on abiological sample, of an HIV-1 subgroup O retrovirus, characterized inthat it comprises as optionally labeled probe, at least one nucleotidesequence according to one of claims 23 to 29 and 34 or a compositionaccording to one of claims 31, 32 or 33, and optionally anothernucleotide probe according to any one of claims 23 to 29 or compositionaccording to any one of claims 31, 32 or 33, which is optionallyimmobilized on a solid support.
 48. Kit according to claim 28,characterized in that it also comprises the reagents required forcarrying out a hybridization.
 49. Process of detection anddiscrimination, in a biological sample, between antibodiescharacteristic of an HIV-1 group (or subgroup) O retrovirus andantibodies characteristic of an HIV-1 subgroup M retrovirus,characterized by the placing in contact of this biological sample with apeptide chosen from peptides (1), (2), (3), (4), (5a) and (5b) of claim8, peptide (9) of claim 10 and peptide (10) of claim
 11. 50. Process ofdetection and discrimination, in a biological sample, between antibodiescharacteristic of an HIV-1 group (or subgroup) O retrovirus andantibodies characteristic of an HIV-1 subgroup M retrovirus,characterized by the placing in contact of this biological sample with apeptide obtained from one of the HIV-1 subgroup M viruses taken intoconsideration in FIGS. 8 and 9 and homologous with a peptide chosen fromthose of claim 49, the sequence of this homologous peptide resultingfrom vertical alignments of its own successive amino acids, which arethemselves contained in the pertinent peptide sequence relative to thecorresponding HIV-1 subgroup M virus and represented in FIG. 8 or 9 withthe successive amino acids of the chosen peptide sequence, as alsofollows from FIG. 8 or
 9. 51. Process of detection and discriminationbetween infection with an HIV-1 group (or subgroup) O retrovirus and ofthe HIV-1 subgroup M type, characterized by the placing in contact ofsera, derived from individuals subjected to a diagnostic test for AIDS,with the peptide RILAVERY.
 52. Process for the detection of infectiondue either to an HIV-1 group (or subgroup) O or HIV-1 subgroup Mretrovirus, characterized by the use of mixtures of two categories ofpeptides, those of the first category corresponding to those identifiedin claim
 49. 53. Process of discrimination between an infection due toan HIV-1 group (or subgroup) O DUR retrovirus or variant, and aninfection due to another type of HIV-1 group (or subgroup) O retrovirus,characterized by the placing in contact of the biological sample studiedwith any one of the following peptides: peptide (11) of claim 38,peptide (12) of claim 39 or peptide (13) of claim 40, peptide (14) orpeptide (15) of claim 41 or peptides (17), (18), (19) and (20) of claim44.
 54. Vector containing a nucleic acid whose nucleotide sequencecorresponds to any one of the sequences of claims 23 to
 30. 55. Vectoraccording to claim 57, characterized in that it is a plasmid. 56.Plasmid chosen from those which were deposited at the CNCM on Feb. 24,1995 under the references I-1548, I-1549 and I-1550.
 57. Cell containinga nucleic acid whose nucleotide sequence corresponds to any one of thesequences of claims 54 and
 55. 58. Virus deposited on Feb. 23, 1995 atthe CNCM under the reference I-1542.
 59. Virus of the same type orsubtype as the virus of claim 58, characterized in that the consensuspeptides of this virus are recognized by antibodies which specificallyrecognize a peptide according to any one of claims 6 to
 22. 60. Kit forthe in vitro detection of antibodies against HIV, containing at leastone peptide according to any one of claims 6 to
 22. 61. Kit according toclaim 60, also containing at least one consensus peptide derived fromanother HIV strain comprising: either an amino acid sequence which isseparate from the sequence of this peptide, in which one or more aminoacids are replaced with other amino acids, with the proviso that thepeptide retains its reactivity with an antiserum against the consensuspeptide, or an amino acid sequence in which one or more amino acids havebeen deleted or added, with the proviso that the peptide or polypeptideretains its reactivity with an antiserum against the consensus peptide.62. Kit according to claim 60 or 61, characterized in that the other HIVstrain is an HIV-LAI strain.
 63. Process of discrimination between aninfection with an HIV-1 group (or subgroup) O retrovirus and an HIV-1subgroup M retrovirus, using a serine protease whose cleaving action iscarried out on an SR dipeptide, and comprising the detection of acleavage or of a on-cleavage of the V3 loop of gp120 of the retrovirus,depending on whether this retrovirus is an HIV-1 group (or subgroup) Oretrovirus or an HIV-1 subgroup M retrovirus.
 64. Viral lysate asobtained by lysis of cells infected with a virus according to claim 58or 59 or with an HIV-1_((VAU)) virus.
 65. Protein extract of HIV-1O_((DUR)) strain containing in particular an antigenic peptide accordingto any one of claims 6 to 22, or of HIV-1 group (or subgroup) O_((VAU))strain containing in particular an antigenic peptide according to anyone of claims 1 to
 5. 66. Bacterial strain deposited at the CCNM on Oct.20, 1994 under the access number I-1486.
 67. Composition for detectionand discrimination, in a biological sample, between an HIV-1 subgroup Mretrovirus and an HIV-1 group (or subgroup) O retrovirus, comprising amixture of two categories of peptides, the first being those identifiedin claim
 49. 68. Peptide according to claim 8, characterized in that itconsists of a peptide whose amino acid sequence is contained either inone of the following sequences: IGGHQGALQ (23) REPTGSDI (24)

or in a corresponding immunologically similar sequence, this peptidecontaining at least 4 consecutive amino acids of one of said sequences.69. Peptide according to claim 7, characterized in that it consists of apeptide whose amino acid sequence is contained in the following aminoacid sequence: INDEAADWD (25) or in a corresponding immunologicallysimilar sequence, this peptide containing at least 4 consecutive aminoacids of said sequence.
 70. Nucleic acid coding for the peptides ofclaims 68 and
 69. 71. Composition comprising at least one nucleic acidaccording to claim
 70. 72. Use of at least one nucleic acid according toclaims 70 and 71 for detection and discrimination between HIV-1 group Mand HIV-1 group O strains.